WT1 HLA class II-binding peptides and compositions and methods comprising same

ABSTRACT

This invention provides WT1 peptides and methods of treating, reducing the incidence of, and inducing immune responses against a WT1-expressing cancer, comprising same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/991,442, filed Jan. 8, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/028,329, filed Sep. 16, 2013, issued as U.S.Pat. No. 9,233,149 which is a continuation of U.S. patent applicationSer. No. 12/083,731, filed Sep. 27, 2009, issued as U.S. Pat. No.8,765,687, which is a National Phase Application of PCT InternationalApplication No. PCT/US06/40719, International Filing Date Oct. 17, 2006,claiming priority to U.S. Provisional Patent Applications, 60/726,608,filed Oct. 17, 2005, and 60/728,304, filed Oct. 20, 2005.

FIELD OF INVENTION

This invention provides WT1 peptides and methods of treating, reducingthe incidence of, and inducing immune responses against a WT1-expressingcancer, comprising same.

BACKGROUND OF THE INVENTION

Wilms tumor (WT), a pediatric nephroblastoma that occurs with afrequency of 1 in 10,000 births, has been the subject of intenseclinical and basic research for several years. The tumor is embryonic inorigin, it is detected in children usually during the first 5 years oflife and can occur unilaterally or bilaterally. A WT arises whencondensed metanephric mesenchymal cells of the developing kidney fail toproperly differentiate. The implication of the Wilms tumor 1 (WT1) tumorsuppressor gene in the etiology of WT illustrated the impact thatgenetic alterations can have on both development and tumorigenesis.

SUMMARY OF THE INVENTION

This invention provides WT1 peptides and methods of treating, reducingthe incidence of, and inducing immune responses against a WT1-expressingcancer, comprising same.

In one embodiment, the present invention provides an isolated WT1peptide having an amino acid (AA) sequence comprising the sequenceRSDELVRHHNMHQRNMTKL (SEQ ID No: 2). In another embodiment, the AAsequence of the isolated WT1 peptide consists of SEQ ID No: 2. Inanother embodiment, the AA sequence of the isolated WT1 consists of afragment of SEQ ID No: 2. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides an isolated WT1peptide having an AA sequence comprising the sequencePGCNKRYFKLSHLQMHSRKHTG (SEQ ID No: 4). In another embodiment, the AAsequence of the isolated WT1 peptide consists of SEQ ID No: 4. Inanother embodiment, the AA sequence of the isolated WT1 consists of afragment of SEQ ID No: 4. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising (a) an antigen-presenting cell and (b) a peptide selectedfrom RSDELVRHHNMHQRNMTKL (SEQ ID No: 2) and PGCNKRYFKLSHLQMHSRKHTG (SEQID No: 4).

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a WT1 vaccine of the present invention,thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a WT1vaccine of the present invention, thereby reducing the incidence of aWT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT1 protein; (b) a fragment of a WTprotein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a fragment of a WT1 protein, therebyinducing an anti-mesothelioma immune response in a subject.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising(a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotidemolecule encoding a WT1 protein; or (d) a nucleotide molecule encoding afragment of a WT1 protein, thereby treating a subject with amesothelioma.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising (a) a WT1 protein; (b) a fragment ofa WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a fragment of a WT1 protein, therebyreducing an incidence of a mesothelioma, or its relapse, in a subject.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E: T2 stabilization assay of native and synthetic WT-1 peptidesto HLA A0201 cells (FIG. 1A) and HLA A0301 cells (FIGS. 1B-E).Fluorescence index is ratio between median fluorescence with peptidetested:median fluorescence with no peptide. X axis: concentration perwell of the peptide tested.

FIGS. 2A-B: CD8+/CD3+ gamma interferon (IFN) ELISPOT (FIG. 2A) andcytotoxicity (FIG. 2B) from healthy HLA A0201 donors against T2 cellspulsed with the following peptides: 1st bar in each series: no peptide;2nd bar: same peptide used for stimulation; 3rd bar: correspondingnative peptide; 4th bar: negative control peptide. X axis: peptides usedfor stimulations. Experiments were performed in triplicate and confirmed3-5 times.

FIGS. 3A-D: CD8+ (FIG. 3A) and CD3+ (FIGS. 3B-D) gamma IFN ELISPOT fromhealthy HLA A0201 donors using the indicated peptides—assignment of barsin each series is the same as for FIG. 2 . Each subfigure in B-Drepresents a separate repetition of the experiment.

FIGS. 4A-B show Cytotoxicity assays using CD8+ T cells stimulated withsynthetic WT-1 A1 peptides from a HLA A0201 donor against HLA-matchedCML blasts presenting native peptide sequences. FIG. 4A. Bar graphs ofresults. 1st bar in each series: SKLY-16 (WT1−); 2nd bar: BV173 (WT1+);3rd bar: LAMA81 (WT1+); 4th bar: CMLA (additional negative control).FIG. 4B. Killing curves. Squares: SKLY-16. Diamonds: 697 cells. G3, F4,C5, and G5 are T-cell clones generated from a healthy HLA-A0201 donorafter multiple stimulations in vitro. Y axis: percentage ofcytotoxicity. X axis: T cell:target cell ratio.

FIGS. 5A-B. FIG. 5A shows gamma interferon ELISPOT after stimulationwith WT1 peptides of CD3+ T cells from healthy donors with differentHLA-DRB1 types. FIG. 5B shows CD3+ T cells (A: HLA-DRB1*1001/1501; B:HLA-DRB1*0701/1202; C: HLA-DRB1*0301/901; D: HLA-DRB*0407/1302) werestimulated twice with peptide WT1DR 328 or WT1DR 423. Stimulated T cellswere challenged in an IFN-gamma ELISPOT assay with the following: GreyBars: unchallenged control; Black Bars: CD14+ cells pulsed withstimulating peptide (either WT1DR 328 or WT1DR 423); White Bars: CD14+cells pulsed with irrelevant CD4+ peptide epitope (RAS); Hatched Bars:unpulsed CD14+ cells. *-p<0.05 compared to controls. Y axis: number ofspots per 1×105 CD3+ T cells. X axis: peptide used for T cellstimulations.

FIGS. 6A-B show peptides of the present invention are processed,presented, and recognized by human T cells. FIG. 6A shows CD3+ T cellsfrom an HLA A0201/301 DRB1*1301/1302 healthy donor were stimulated withautologous DCs previously incubated with 697 tumor lysates, thenchallenged in an IFN-gamma ELISPOT assay with autologous DCs previouslyincubated with either 697 tumor lysate, individual WT1 peptides, controlpeptides or unpulsed DCs (X axis). FIG. 6B. CD3+ T cells from an HLAA0201/101, DRB1*0301/1601 healthy donor were stimulated with autologousDCs previously incubated with tumor lysates from either JMN (BlackBars), or MeWo (White Bars). T cells were challenged in an IFN-gammaELISPOT assay with autologous DCs previously incubated with JMN or MeWotumor lysates, individual WT1DR peptides, or control class II peptide (Xaxis). Hatched bars: background level of spots from autologous DCsincubated in the absence of T cells. *-P<0.05 compared to controlpeptides. Y axis: number of spots per 1×105 CD3+ cells.

FIG. 7 shows CD3+ gamma interferon ELISPOT against Mesothelioma celllines. Total PBMCs from an HLA-A0201 donor were stimulated twice withthe different WT1DR peptides, then T cells were challenged in anIFN-gamma ELISPOT assay with the following: Mesothelioma H-Meso1A cellline (Black Bars; WT1+, A0201+); control melanoma MeWo cell line (WT1−,A0201+; Grey Bars). *-p≤0.01 compared to MeWo controls. Y axis: numberof spots per 2×10⁵ PBMCs. X axis: peptide used for T cell stimulation.

FIG. 8 shows CD3⁺ gamma IFN ELISPOT against Mesothelioma cell lines.Total PBMCs from an HLA-A0201 donor were stimulated twice with thedifferent WT1DR peptides, then T cells were challenged in an IFN-gammaELISPOT assay with the following: Mesothelioma H-Meso1A cell line (BlackBars; WT1+, A0201+); MeWo cell line (WT1, A0201+; Grey Bars). *-p≤0.01compared to MeWo controls. Y axis: number of spots per 2×10⁵ PBMCs. Xaxis: peptide used for T cell stimulation.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides WT1 peptides and methods of treating, reducingthe incidence of, and inducing immune responses against a WT1-expressingcancer, comprising same.

As provided herein, peptides of the present invention elicit CD4⁺ T cellresponses (Examples 3-4).

In one embodiment, the present invention provides an isolated WT1peptide having an amino acid (AA) sequence comprising the sequenceRSDELVRHHNMHQRNMTKL (SEQ ID No: 2). In another embodiment, the AAsequence of the isolated WT1 peptide consists of SEQ ID No: 2. Inanother embodiment, the AA sequence of the isolated WT1 consists of afragment of SEQ ID No: 2. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides an isolated WT1peptide having an AA sequence comprising the sequencePGCNKRYFKLSHLQMHSRKHTG (SEQ ID No: 4). In another embodiment, the AAsequence of the isolated WT1 peptide consists of SEQ ID No: 4. Inanother embodiment, the AA sequence of the isolated WT1 consists of afragment of SEQ ID No: 4. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides an isolated WT1peptide having an AA sequence comprising or consisting of SEQ ID No: 1,or consisting of a fragment of SEQ ID No: 1. In another embodiment, thepresent invention provides an isolated WT1 peptide having an AA sequencecomprising or consisting of SEQ ID No: 3, or consisting of a fragment ofSEQ ID No: 3. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, an isolated WT1 peptide of the present inventionis unaltered (e.g. its sequence corresponds to a fragment of the WT1protein, without in vitro introduction of mutations.

In another embodiment, the present invention provides a compositioncomprising (a) an antigen-presenting cell and (b) a peptide selectedfrom RSDELVRHHNMHQRNMTKL (SEQ ID No: 2) and PGCNKRYFKLSHLQMHSRKHTG (SEQID No: 4). In another embodiment, the composition further comprises anadditional HLA class II molecule-binding peptide. In another embodiment,the composition further comprise an HLA class I molecule-binding WT1peptide. In another embodiment, the HLA class I molecule is an HLA-Amolecule. In another embodiment, the AA sequence of the HLA class Imolecule-binding WT1 peptide comprises a sequence selected from SEQ IDNo: 5-38. In another embodiment, the AA sequence of the HLA class Imolecule-binding WT1 peptide is from SEQ ID No: 5-38. Each possibilityrepresents a separate embodiment of the present invention.

The WT1 protein of methods and compositions of the present invention canbe any WT1 protein known in the art.

The WT1 molecule from which a peptide of the present invention isderived has, in another embodiment, the sequence:

(GenBank Accession number AY245105; SEQ ID No: 50)MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRN MTKLQLAL.

In another embodiment, the WT1 molecule has the sequence:

(GenBank Accession number NM_000378; SEQ ID No: 51)AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNM HQRNMTKLQLAL.

In another embodiment, the WT1 molecule has the sequence:

(GenBank Accession number NP_077742; SEQ ID No: 52)MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTPDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL.

In another embodiment, the WT1 molecule comprises the sequence:

(SEQ ID No: 53) MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARS DELVRHHNMHQRNMTKLQLAL.

In another embodiment, the WT1 protein has the sequence set forth inGenBank Accession #NM_024426. In other embodiments, the WT1 protein hasor comprises one of the sequences set forth in 1 of the followingsequence entries: NM_024425, NM_024424, NM_000378, S95530, D13624,D12496, D12497, or X77549. In another embodiment, the WT1 protein hasany other WT1 sequence known in the art.

“Peptide,” in another embodiment of methods and compositions of thepresent invention, refers to a compound of subunit AA connected bypeptide bonds. In another embodiment, the peptide comprises an AAanalogue. In another embodiment, the peptide comprises a peptidomimetic.The different AA analogues and peptidomimetics that can be included inthe peptides of methods and compositions of the present invention areenumerated hereinbelow. The subunits are, in another embodiment, linkedby peptide bonds. In another embodiment, the subunit is linked byanother type of bond, e.g. ester, ether, etc. Each possibilityrepresents a separate embodiment of the present invention.

The unaltered and heteroclitic WT1 peptides of the present invention (asdescribed both above and below) are referred to collectively herein as“WT1 peptides.” Each of the embodiments enumerated below for “WT1peptides” applies to unaltered WT1 peptides and HLA class I and class IIheteroclitic peptides of the present invention. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds toan HLA class II molecule. In another embodiment, the HLA class IImolecule is an HLA-DRB molecule. In another embodiment, the HLA classII-molecule is an HLA-DRA molecule. In another embodiment, the HLAmolecule is an HLA-DQA1 molecule. In another embodiment, the HLAmolecule is an HLA-DQB1 molecule. In another embodiment, the HLAmolecule is an HLA-DPA1 molecule. In another embodiment, the HLAmolecule is an HLA-DPB1 molecule. In another embodiment, the HLAmolecule is an HLA-DMA molecule. In another embodiment, the HLA moleculeis an HLA-DMB molecule. In another embodiment, the HLA molecule is anHLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOBmolecule. In another embodiment, the HLA molecule is any other HLA classII-molecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT1 peptide of methods and compositions of thepresent invention is so designed as to exhibit affinity for an HLAmolecule. In another embodiment, the affinity is a high affinity, asdescribed herein.

HLA molecules, known in another embodiment as major histocompatibilitycomplex (MHC) molecules, bind peptides and present them to immune cells.Thus, in another embodiment, the immunogenicity of a peptide ispartially determined by its affinity for HLA molecules. HLA class Imolecules interact with CD8 molecules, which are generally present oncytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4molecules, which are generally present on helper T lymphocytes.

In another embodiment, a peptide of the present invention isimmunogenic. In another embodiment, “immunogenic” refers to an abilityto stimulate, elicit or participate in an immune response. In anotherembodiment, the immune response elicited is a cell-mediated immuneresponse. In another embodiment, the immune response is a combination ofcell-mediated and humoral responses.

In another embodiment, T cells that bind to the MHC molecule-peptidecomplex become activated and induced to proliferate and lyse cellsexpressing a protein comprising the peptide. T cells are typicallyinitially activated by “professional” antigen presenting cells (“APC”;e.g. dendritic cells, monocytes, and macrophages), which presentcostimulatory molecules that encourage T cell activation as opposed toanergy or apoptosis. In another embodiment, the response isheteroclitic, as described herein, such that the CTL lyses a neoplasticcell expressing a protein which has an AA sequence homologous to apeptide of this invention, or a different peptide than that used tofirst stimulate the T cell.

In another embodiment, an encounter of a T cell with a peptide of thisinvention induces its differentiation into an effector and/or memory Tcell. Subsequent encounters between the effector or memory T cell andthe same peptide, or, in another embodiment, with a related peptide ofthis invention, leads to a faster and more intense immune response. Suchresponses are gauged, in another embodiment, by measuring the degree ofproliferation of the T cell population exposed to the peptide. Inanother embodiment, such responses are gauged by any of the methodsenumerated hereinbelow.

In another embodiment, the peptides of methods and compositions of thepresent invention bind an HLA class II molecule with high affinity. Inother embodiments, the HLA class II molecule is any HLA class IImolecule enumerated herein. Each possibility represents a separateembodiment of the present invention.

In another embodiment, derivatives of peptides of methods andcompositions of the present invention bind an HLA class I molecule withhigh affinity. In other embodiments, the MHC class I molecule is any MHCclass I molecule enumerated herein. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention binds an HLA class II molecule with significantaffinity, while a peptide derived from the original peptide binds an HLAclass I molecule with significant affinity.

In another embodiment, “affinity” refers to the concentration of peptidenecessary for inhibiting binding of a standard peptide to the indicatedMHC molecule by 50%. In another embodiment, “high affinity” refers to anaffinity is such that a concentration of about 500 nanomolar (nM) orless of the peptide is required for 50% inhibition of binding of astandard peptide. In another embodiment, a concentration of about 400 nMor less of the peptide is required. In another embodiment, the bindingaffinity is 300 nM. In another embodiment, the binding affinity is 200nM. In another embodiment, the binding affinity is 150 nM. In anotherembodiment, the binding affinity is 100 nM. In another embodiment, thebinding affinity is 80 nM. In another embodiment, the binding affinityis 60 nM. In another embodiment, the binding affinity is 40 nM. Inanother embodiment, the binding affinity is 30 nM. In anotherembodiment, the binding affinity is 20 nM. In another embodiment, thebinding affinity is 15 nM. In another embodiment, the binding affinityis 10 nM. In another embodiment, the binding affinity is 8 nM. Inanother embodiment, the binding affinity is 6 nM. In another embodiment,the binding affinity is 4 nM. In another embodiment, the bindingaffinity is 3 nM. In another embodiment, the binding affinity is 2 nM.In another embodiment, the binding affinity is 1.5 nM. In anotherembodiment, the binding affinity is 1 nM. In another embodiment, thebinding affinity is 0.8 nM. In another embodiment, the binding affinityis 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. Inanother embodiment, the binding affinity is 0.4 nM. In anotherembodiment, the binding affinity is 0.3 nM. In another embodiment, thebinding affinity is less than 0.3 nM.

In another embodiment, “affinity” refers to a measure of bindingstrength to the MHC molecule. In another embodiment, affinity ismeasured using a method known in the art to measure competitive bindingaffinities. In another embodiment, affinity is measured using a methodknown in the art to measure relative binding affinities. In anotherembodiment, the method is a competitive binding assay. In anotherembodiment, the method is radioimmunoassay or RIA. In anotherembodiment, the method is BiaCore analyses. In another embodiment, themethod is any other method known in the art. In another embodiment, themethod yields an IC50 in relation to an IC50 of a reference peptide ofknown affinity.

Each type of affinity and method of measuring affinity represents aseparate embodiment of the present invention.

In another embodiment, “high affinity” refers to an IC50 of 0.5-500 nM.In another embodiment, the IC50 is 1-300 nM. In another embodiment, theIC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. Inanother embodiment, the IC50 is 3-100 nM. In another embodiment, theIC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. Inanother embodiment, the IC50 is 10-100 nM. In another embodiment, theIC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. Inanother embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In anotherembodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is10-50 nM. In another embodiment, the IC50 is 20-50 nM. In anotherembodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment,the IC50 is 15-25 nM. Each affinity and range of affinities represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention binds to a superfamily of HLA molecules. Superfamiliesof HLA molecules share very similar or identical binding motifs. Inanother embodiment, the superfamily is a HLA class I superfamily. Inanother embodiment, the superfamily is a HLA class II superfamily. Eachpossibility represents a separate embodiment of the present invention.

The terms “HLA-binding peptide,” “HLA class I molecule-binding peptide,”and “HLA class II molecule-binding peptide” refer, in anotherembodiment, to a peptide that binds an HLA molecule with measurableaffinity. In another embodiment, the terms refer to a peptide that bindsan HLA molecule with high affinity. In another embodiment, the termsrefer to a peptide that binds an HLA molecule with sufficient affinityto activate a T cell precursor. In another embodiment, the terms referto a peptide that binds an HLA molecule with sufficient affinity tomediate recognition by a T cell. The HLA molecule is, in otherembodiments, any of the HLA molecules enumerated herein. Eachpossibility represents a separate embodiment of the present invention.

“Heteroclitic” refers, in another embodiment, to a peptide thatgenerates an immune response that recognizes the original peptide fromwhich the heteroclitic peptide was derived (e.g. the peptide notcontaining the anchor residue mutations). In another embodiment,“original peptide” refers to a peptide of the present invention. Forexample, YMFPNAPYL (SEQ ID No: 6), was generated from RMFPNAPYL (SEQ IDNo: 5) by mutation of residue 1 to tyrosine (Examples). In anotherembodiment, “heteroclitic” refers to a peptide that generates an immuneresponse that recognizes the original peptide from which theheteroclitic peptide was derived, wherein the immune response generatedby vaccination with the heteroclitic peptide is greater than the immuneresponse generated by vaccination with the original peptide. In anotherembodiment, a “heteroclitic” immune response refers to an immuneresponse that recognizes the original peptide from which the improvedpeptide was derived (e.g. the peptide not containing the anchor residuemutations). In another embodiment, a “heteroclitic” immune responserefers to an immune response that recognizes the original peptide fromwhich the heteroclitic peptide was derived, wherein the magnitude of theimmune response generated by vaccination with the heteroclitic peptideis greater than the immune response generated by vaccination with theoriginal peptide. In another embodiment, the magnitude of the immuneresponse generated by vaccination with the heteroclitic peptide isgreater than the immune response substantially equal to the response tovaccination with the original peptide. In another embodiment, themagnitude of the immune response generated by vaccination with theheteroclitic peptide is greater than the immune response less than theresponse to vaccination with the original peptide. In anotherembodiment, a heteroclitic peptide of the present invention is an HLAclass I heteroclitic peptide. Methods for identifying HLA class I andclass II residues, and for improving HLA binding by mutating theresidues, are well known in the art, as described below. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a heteroclitic peptide of the present inventioninduces an immune response that is increased at least 2-fold relative tothe WT1 peptide from which the heteroclitic peptide was derived (“nativepeptide”). In another embodiment, the increase is 3-fold relative to thenative peptide. In another embodiment, the increase is 5-fold relativeto the native peptide. In another embodiment, the increase is 7-foldrelative to the native peptide. In another embodiment, the increase is10-fold relative to the native peptide. In another embodiment, theincrease is 15-fold relative to the native peptide. In anotherembodiment, the increase is 20-fold relative to the native peptide. Inanother embodiment, the increase is 30-fold relative to the nativepeptide. In another embodiment, the increase is 50-fold relative to thenative peptide. In another embodiment, the increase is 100-fold relativeto the native peptide. In another embodiment, the increase is 150-foldrelative to the native peptide. In another embodiment, the increase is200-fold relative to the native peptide. In another embodiment, theincrease is 300-fold relative to the native peptide. In anotherembodiment, the increase is 500-fold relative to the native peptide. Inanother embodiment, the increase is 1000-fold relative to the nativepeptide. In another embodiment, the increase is more than 1000-foldrelative to the native peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a HLA class IIheteroclitic peptide derived from an isolated WT1 peptide of the presentinvention. In another embodiment, the process of deriving comprisesintroducing a mutation that enhances a binding of the peptide to an HLAclass II molecule. In another embodiment, the process of derivingconsists of introducing a mutation that enhances a binding of thepeptide to an HLA class I molecule. In another embodiment, the mutationis in an HLA class II anchor residue. In another embodiment, aheteroclitic class II peptide of the present invention is identified andtested in a manner analogous to identification and testing of HLA classI heteroclitic peptides, as exemplified herein. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the HLA class II binding site in a peptide of thepresent invention is created or improved by mutation of an HLA class IImotif anchor residue. In another embodiment, the anchor residue that ismodified is in the P1 position. In another embodiment, the anchorresidue is at the P2 position. In another embodiment, the anchor residueis at the P6 position. In another embodiment, the anchor residue is atthe P9 position. In another embodiment, the anchor residue is selectedfrom the P1, P2, P6, and P9 positions. In another embodiment, the anchorresidue is at the P3 position. In another embodiment, the anchor residueis at the P4 position. In another embodiment, the anchor residue is atthe P5 position. In another embodiment, the anchor residue is at the P6position. In another embodiment, the anchor residue is at the P8position. In another embodiment, the anchor residue is at the P10position. In another embodiment, the anchor residue is at the P11position. In another embodiment, the anchor residue is at the P12position. In another embodiment, the anchor residue is at the P13position. In another embodiment, the anchor residue is at any otheranchor residue of an HLA class II molecule that is known in the art. Inanother embodiment, residues other than P1, P2, P6, and P9 serve assecondary anchor residues; therefore, mutating them can improve HLAclass II binding. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, a heteroclitic peptide is generated byintroduction of a mutation that creates an anchor motif. “Anchor motifs”or “anchor residues” refers, in another embodiment, to 1 or a set ofpreferred residues at particular positions in an HLA-binding sequence.In another embodiment, the HLA-binding sequence is an HLA classII-binding sequence. In another embodiment, the HLA-binding sequence isan HLA class I-binding sequence. In another embodiment, the positionscorresponding to the anchor motifs are those that play a significantrole in binding the HLA molecule. In another embodiment, the anchorresidue is a primary anchor motif. In another embodiment, the anchorresidue is a secondary anchor motif. Each possibility represents aseparate embodiment of the present invention.

Methods for predicting MHC class II epitopes are well known in the art.In another embodiment, the MHC class II epitope is predicted usingTEPITOPE (Meister G E, Roberts C G et al, Vaccine 1995 13: 581-91). Inanother embodiment, the MHC class II epitope is predicted usingEpiMatrix (De Groot A S, Jesdale B M et al, AIDS Res Hum Retroviruses1997 13: 529-31). In another embodiment, the MHC class II epitope ispredicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med.2002 8: 137-48). In another embodiment, the MHC class II epitope ispredicted using the SYFPEITHI epitope prediction algorithm (Examples).In another embodiment, the MHC class II epitope is predicted usingRankpep. In another embodiment, the MHC class II epitope is predictedusing any other method known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, in the case of HLA class II-binding peptides(e.g. HLA-DR-binding peptides), the anchor residue that is modified isin the P1 position (e.g. a position corresponding to F263 of theCII(259-273) peptide, an unrelated peptide that was used to define someof the anchor residues of an HLA-DR allele). In another embodiment, theanchor residue is in the P2 position (e.g. a position corresponding toK264 of the CII(259-273) peptide). In another embodiment, the anchorresidue is in the P6 position. In another embodiment, the anchor residueis in the P9 position. In other embodiments, the anchor residue is theP3, P4, P5, P6, P8, P10, P11, P12, or P13 position. In anotherembodiment, the anchor residue is any other anchor residue of an HLAclass II molecule that is known in the art. In another embodiment,residues other than P1, P2, P6, and P9 serve as secondary anchorresidues; therefore, mutating them can improve HLA class II binding. Inanother embodiment, any combination of the above residues is mutated.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a WT1 peptide of the present invention binds to 2distinct HLA class II molecules. In another embodiment, the peptidebinds to three distinct HLA class II molecules. In another embodiment,the peptide binds to four distinct HLA class II molecules. In anotherembodiment, the peptide binds to five distinct HLA class II molecules.In another embodiment, the peptide binds to six distinct HLA class IImolecules. In another embodiment, the peptide binds to more than sixdistinct HLA class II molecules.

In another embodiment, the HLA class II molecules that are bound by aWT1 peptide of the present invention are encoded by two or more distinctalleles at a given HLA class II locus. In another embodiment, the HLAclass II molecules are encoded by 3 distinct alleles at a locus. Inanother embodiment, the HLA class II molecules are encoded by 4 distinctalleles at a locus. In another embodiment, the HLA class II moleculesare encoded by 5 distinct alleles at a locus. In another embodiment, theHLA class II molecules are encoded by 6 distinct alleles at a locus. Inanother embodiment, the HLA class II molecules are encoded by more thansix distinct alleles at a locus.

In another embodiment, the HLA class II molecules bound by the WT1peptide are encoded by HLA class II genes at 2 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at2 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at 3 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at3 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at 4 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at4 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at more than 4 distinct loci. In otherembodiments, the loci are selected from HLA-DRB loci. In anotherembodiment, the HLA class II-binding peptide is an HLA-DRA bindingpeptide. In another embodiment, the peptide is an HLA-DQA1 bindingpeptide. In another embodiment, the peptide is an HLA-DQB1 bindingpeptide. In another embodiment, the peptide is an HLA-DPA1 bindingpeptide. In another embodiment, the peptide is an HLA-DPB1 bindingpeptide. In another embodiment, the peptide is an HLA-DMA bindingpeptide. In another embodiment, the peptide is an HLA-DMB bindingpeptide. In another embodiment, the peptide is an HLA-DOA bindingpeptide. In another embodiment, the peptide is an HLA-DOB bindingpeptide. In another embodiment, the peptide binds to any other HLA classII molecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds to 2distinct HLA-DRB molecules. In another embodiment, the peptide binds to3 distinct HLA-DRB molecules. In another embodiment, the peptide bindsto 4 distinct HLA-DRB molecules. In another embodiment, the peptidebinds to 5 distinct HLA-DRB molecules. In another embodiment, thepeptide binds to 6 distinct HLA-DRB molecules. In another embodiment,the peptide binds to more than 6 distinct HLA-DRB molecules.

In another embodiment, a WT1 peptide of the present invention binds toHLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles. Inanother embodiment, the HLA-DRB molecules are encoded by 3 distinctHLA-DRB alleles. In another embodiment, the HLA-DRB molecules areencoded by 4 distinct HLA-DRB alleles. In another embodiment, theHLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles. In anotherembodiment, the HLA-DRB molecules are encoded by 6 distinct HLA-DRBalleles. In another embodiment, the HLA-DRB molecules are encoded bymore than 6 distinct HLA-DRB alleles. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds toHLA-DRB molecules that are encoded by 2 distinct HLA-DRB allelesselected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB1501. In another embodiment, the WT1 peptide binds to HLA-DRB moleculesencoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301,DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRB allelesselected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB1501. In another embodiment, the WT1 peptide binds to HLA-DRB moleculesencoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301,DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1peptide binds to HLA-DRB molecules encoded by each of the followingHLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB1501. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a compositioncomprising 2 distinct WT1 peptides of the present invention. In anotherembodiment, the 2 distinct WT1 peptides are both unaltered. In anotherembodiment, 1 of the WT1 peptides is unaltered, while the other isheteroclitic. In another embodiment, both of the WT1 peptides areheteroclitic.

In another embodiment, the composition comprises 3 distinct WT1 peptidesof the present invention. In another embodiment, the compositioncomprises 4 distinct WT1 peptides of the present invention. In anotherembodiment, the composition comprises 5 distinct WT1 peptides of thepresent invention. In another embodiment, the composition comprises morethan 5 distinct isolated WT1 peptides of the present invention.

In another embodiment, 2 of the WT1 peptides in the composition areunaltered. In another embodiment, 2 of the WT1 peptides in thecomposition are heteroclitic. In another embodiment, 2 of the WT1peptides in the composition are unaltered, and 2 are heteroclitic. Inanother embodiment, more than 2 of the WT1 peptides in the compositionare unaltered. In another embodiment, more than 2 of the WT1 peptides inthe composition are heteroclitic. In another embodiment, more than 2 ofthe WT1 peptides in the composition are unaltered, and more than 2 areheteroclitic. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, 1 of the additional WT1 peptides in a compositionof the present invention has a sequence selected from the sequences setforth in SEQ ID No: 1-3. In another embodiment, 2 of the additional WT1peptides have a sequence selected from the sequences set forth in SEQ IDNo: 1-3. In another embodiment, 3 of the additional WT1 peptides have asequence selected from the sequences set forth in SEQ ID No: 1-3.

In another embodiment, any other immunogenic WT1 peptide known in theart is utilized as an additional WT1 peptide. In another embodiment, anycombination of immunogenic WT1 peptides known in the art is utilized.

Each additional WT1 peptide, and each combination thereof, represents aseparate embodiment of the present invention.

In another embodiment, a composition of the present invention contains 2HLA class II heteroclitic peptides that are derived from the sameisolated WT1 peptide of the present invention. In another embodiment,the 2 HLA class II heteroclitic peptides contain mutations in differentHLA class II molecule anchor residues. In another embodiment, the 2 HLAclass II heteroclitic peptides contain different mutations in the sameanchor residues. In another embodiment, 2 of the HLA class IIheteroclitic peptides are derived from different isolated WT1 peptidesof the present invention. Each possibility represents a separateembodiment of the present invention.

In another embodiment, 2 WT1 peptides of the present invention, or theWT1 peptides that correspond to two HLA class II heteroclitic peptidesof the present invention, overlap with one another. In anotherembodiment, the overlap between the peptides is at least 7 amino acids(AA). In another embodiment, the overlap is at least 8 AA. In anotherembodiment, the overlap is at least 9 AA. In another embodiment, theoverlap is 7 AA. In another embodiment, the overlap is 8 AA. In anotherembodiment, the overlap is 9 AA. In another embodiment, the overlap is10 AA. In another embodiment, the overlap is 11 AA. In anotherembodiment, the overlap is 12 AA. In another embodiment, the overlap is13 AA. In another embodiment, the overlap is 14 AA. In anotherembodiment, the overlap is 15 AA. In another embodiment, the overlap is16 AA. In another embodiment, the overlap is more than 16 AA. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the peptides in a composition of the presentinvention bind to 2 distinct HLA class II molecules. In anotherembodiment, the peptides bind to 3 distinct HLA class II molecules. Inanother embodiment, the peptides bind to 4 distinct HLA class IImolecules. In another embodiment, the peptides bind to 5 distinct HLAclass II molecules. In another embodiment, the peptides bind to morethan 5 distinct HLA class II molecules. In another embodiment, thepeptides in the composition bind to the same HLA class II molecules.

In another embodiment, each of the WT1 peptides in a composition of thepresent invention binds to a set of HLA class II molecules. In anotherembodiment, each of the WT1 peptides binds to a distinct set of HLAclass II molecules. In another embodiment, the WT1 peptides in thecomposition bind to the same set of HLA class II molecules. In anotherembodiment, 2 of the WT1 peptides bind to a distinct but overlapping setof HLA class II molecules. In another embodiment, 2 or more of the WT1peptides bind to the same set of HLA class II molecules, while anotherof the WT1 peptides binds to a distinct set. In another embodiment, 2 ormore of the WT1 peptides bind to an overlapping set of HLA class IImolecules, while another of the WT1 peptides binds to a distinct set.

In another embodiment, 2 or more of the WT1 peptides in a composition ofthe present invention each binds to more than 1 HLA-DRB molecule. Inanother embodiment, the 4 or more HLA-DRB molecules bound by thepeptides in the composition are distinct from one another. In anotherembodiment, the HLA-DRB molecules are encoded by different HLA-DRBalleles. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, 2 or more of the HLA class II molecules bound byWT1 peptides in a composition of the present invention are HLA-DRBmolecules. In another embodiment, 3 or more of the HLA class IImolecules that are bound are HLA-DRB molecules. In other embodiments,the HLA class II molecules that are bound can be any of the HLA class IImolecules enumerated herein. In another embodiment, the HLA class IImolecules that are bound are encoded by 2 or more distinct HLA class IIalleles at a given locus. In another embodiment, the HLA class IImolecules that are bound are encoded by HLA class II genes at 2 or moredistinct loci.

Each of the above compositions represents a separate embodiment of thepresent invention.

In another embodiment, a “set of HLA class II molecules” refers to theHLA class II molecules encoded by different alleles at a particularlocus. In another embodiment, the term refers to HLA class II moleculeswith a particular binding specificity. In another embodiment, the termrefers to HLA class II molecules with a particular peptide consensussequence. In another embodiment, the term refers to a superfamily of HLAclass II molecules. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a compositioncomprising an unaltered HLA class II molecule-binding WT1 peptide of thepresent invention and a second, HLA class I molecule-binding WT1peptide. In another embodiment, the composition comprises more than 1HLA class II molecule-binding WT1 peptide of the present invention, inaddition to the HLA class I molecule-binding WT1 peptide. In anotherembodiment, the composition comprises more than 1 HLA class Imolecule-binding WT1 peptide, in addition to the HLA class IImolecule-binding WT1 peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the AA sequence of the HLA class Imolecule-binding WT1 peptide comprises a sequence selected from SEQ IDNo: 5-38. In another embodiment, the AA sequence of the HLA class Imolecule-binding WT1 peptide is selected from the sequences set forth inSEQ ID No: 5-38. Each possibility represents a separate embodiment ofthe present invention.

In other embodiments, the HLA class I molecule bound by the HLA class Imolecule-binding WT1 peptide is encoded by any of the HLA-A genes. Inother embodiments, the HLA class I molecule is encoded by any of theHLA-B genes. In other embodiments, the HLA class I molecule is encodedby any of the HLA-C genes. In another embodiment, the HLA class Imolecule is an HLA-0201 molecule. In another embodiment, the molecule isHLA A1. In other embodiments, the molecule is HLA A3.2, HLA A11, HLAA24, HLA B7, HLA B8, HLA B27, or HLA A2, A3, A4, A5, or B8. HLA A1, HLAA2.1, or HLA A3.2. In other embodiment, the HLA class II molecule isencoded by any of the HLA genes HLA-DP, -DQ, or -DR. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the HLA class I molecule-binding WT1 peptide ofmethods and compositions of the present invention binds to a superfamilyof HLA class I molecules. In another embodiment, the superfamily is theA2 superfamily. In another embodiment, the superfamily is the A3superfamily. In another embodiment, the superfamily is the A24superfamily. In another embodiment, the superfamily is the B7superfamily. In another embodiment, the superfamily is the B27superfamily. In another embodiment, the superfamily is the B44superfamily. In another embodiment, the superfamily is the C1superfamily. In another embodiment, the superfamily is the C4superfamily. In another embodiment, the superfamily is any othersuperfamily known in the art. Each possibility represents a separateembodiment of the present invention. In another embodiment, the HLAmolecule is HLA A0201.

In another embodiment, the HLA class I molecule-binding WT1 peptide isan HLA class I heteroclitic peptide. In another embodiment, the HLAclass I molecule-binding WT1 peptide contains a mutation in an HLA classI molecule anchor residue thereof, as described further herein. Asprovided herein, WT1-derived peptides were modified in HLA anchorresidues to generate heteroclitic peptides with increased predictedbinding to HLA-A0201 and HLA-A0301. Peptides with increased predictedbinding also exhibited enhanced ability to bind HLA class I moleculesand increased immunogenicity.

In another embodiment, the mutation that enhances MHC binding is in theresidue at position 1 of the HLA class I heteroclitic peptide. Inanother embodiment, the residue is changed to tyrosine. In anotherembodiment, the residue is changed to glycine. In another embodiment,the residue is changed to threonine. In another embodiment, the residueis changed to phenylalanine. In another embodiment, the residue ischanged to any other residue known in the art. In another embodiment, asubstitution in position 1 (e.g. to tyrosine) stabilizes the binding ofthe position 2 anchor residue.

In another embodiment, the mutation is in position 2 of the HLA class Iheteroclitic peptide. In another embodiment, the residue is changed toleucine. In another embodiment, the residue is changed to valine. Inanother embodiment, the residue is changed to isoleucine. In anotherembodiment, the residue is changed to methionine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the mutation is in position 6 of the HLA class Iheteroclitic peptide. In another embodiment, the residue is changed tovaline. In another embodiment, the residue is changed to cysteine. Inanother embodiment, the residue is changed to glutamine. In anotherembodiment, the residue is changed to histidine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the mutation is in position 9 of the HLA class Iheteroclitic peptide. In another embodiment, the mutation changes theresidue at the C-terminal position thereof. In another embodiment, theresidue is changed to valine. In another embodiment, the residue ischanged to threonine. In another embodiment, the residue is changed toisoleucine. In another embodiment, the residue is changed to leucine. Inanother embodiment, the residue is changed to alanine. In anotherembodiment, the residue is changed to cysteine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the point mutation is in a primary anchorresidue. In another embodiment, the HLA class I primary anchor residuesare positions 2 and 9. In another embodiment, the point mutation is in asecondary anchor residue. In another embodiment, the HLA class Isecondary anchor residues are positions 1 and 8. In another embodiment,the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and8. In another embodiment, the point mutation is in a position selectedfrom positions 4, 5, and 8. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the point mutation is in 1 or more residues inpositions selected from positions 1, 2, 8, and 9 of the HLA class Ibinding motif. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 1, 3, 6, and 9. In anotherembodiment, the point mutation is in 1 or more residues in positionsselected from positions 1, 2, 6, and 9. In another embodiment, the pointmutation is in 1 or more residues in positions selected from positions1, 6, and 9. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 1, 2, and 9. In anotherembodiment, the point mutation is in 1 or more residues in positionsselected from positions 1, 3, and 9. In another embodiment, the pointmutation is in 1 or more residues in positions selected from positions 2and 9. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 6 and 9. Each possibilityrepresents a separate embodiment of the present invention.

Each of the above anchor residues and substitutions represents aseparate embodiment of the present invention.

In another embodiment, the HLA class I molecule-binding WT1 peptidecomprises a sequence selected from SEQ ID No: 6, 8, 10, 12, 14, 16, 18,20, 22, 24-26, 28-30, 32-34, and 36-38. In another embodiment, the HLAclass I molecule-binding WT1 peptide has a sequence selected from thesequences set forth in SEQ ID No: 6, 8, 10, 12, 14, 16, 18, 20, 22,24-26, 28-30, 32-34, and 36-38.

In another embodiment, the HLA class I molecule-binding WT peptide has alength of 9-13 AA. In another embodiment, the length is 8-13 AA. Inanother embodiment, the peptide has any of the lengths of a peptide ofthe present invention enumerated herein.

In another embodiment, the HLA class I molecule-binding WT peptide haslength of 9 AA. In another embodiment, the peptide has length of 10 AA.As provided herein, native and heteroclitic peptides of 9-10 AAexhibited substantial binding to HLA class I molecules and ability toelicit cytokine secretion and cytolysis by CTL.

In another embodiment, the HLA class I molecule that is bound by the HLAclass I molecule-binding WT1 peptide is an HLA-A molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A2 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A3 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A11 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-B8 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-0201 molecule. In anotherembodiment, the HLA class I-molecule binds any other HLA class Imolecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT1 peptide of methods and compositions of thepresent invention has a length of 8-30 amino acids. In anotherembodiment, the peptide has a length of 9-11 AA. In another embodiment,the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11,or in another embodiment, 8-15, or in another embodiment, 9-20, or inanother embodiment, 9-18, or in another embodiment, 9-15, or in anotherembodiment, 8-12, or in another embodiment, 9-11 AA in length. Inanother embodiment, the peptide is 8 AA in length, or in anotherembodiment, 9 AA or in another embodiment, 10 AA or in anotherembodiment, 12 AA or in another embodiment, 25 AA in length, or inanother embodiment, any length therebetween. In another embodiment, thepeptide is of greater length, for example 50, or 100, or more. In thisembodiment, the cell processes the peptide to a length of 7 and 25 AA inlength. In this embodiment, the cell processes the peptide to a lengthof 9-11 AA Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the peptide is 15-23 AA in length. In anotherembodiment, the length is 15-24 AA. In another embodiment, the length is15-25 AA. In another embodiment, the length is 15-26 AA. In anotherembodiment, the length is 15-27 AA. In another embodiment, the length is15-28 AA. In another embodiment, the length is 14-30 AA. In anotherembodiment, the length is 14-29 AA. In another embodiment, the length is14-28 AA. In another embodiment, the length is 14-26 AA. In anotherembodiment, the length is 14-24 AA. In another embodiment, the length is14-22 AA. In another embodiment, the length is 14-20 AA. In anotherembodiment, the length is 16-30 AA. In another embodiment, the length is16-28 AA. In another embodiment, the length is 16-26 AA. In anotherembodiment, the length is 16-24 AA. In another embodiment, the length is16-22 AA. In another embodiment, the length is 18-30 AA. In anotherembodiment, the length is 18-28 AA. In another embodiment, the length is18-26 AA. In another embodiment, the length is 18-24 AA. In anotherembodiment, the length is 18-22 AA. In another embodiment, the length is18-20 AA. In another embodiment, the length is 20-30 AA. In anotherembodiment, the length is 20-28 AA. In another embodiment, the length is20-26 AA. In another embodiment, the length is 20-24 AA. In anotherembodiment, the length is 22-30 AA. In another embodiment, the length is22-28 AA. In another embodiment, the length is 22-26 AA. In anotherembodiment, the length is 24-30 AA. In another embodiment, the length is24-28 AA. In another embodiment, the length is 24-26 AA.

Each of the above peptides, peptide lengths, and types of peptidesrepresents a separate embodiment of the present invention.

In another embodiment, minor modifications are made to peptides of thepresent invention without decreasing their affinity for HLA molecules orchanging their TCR specificity, utilizing principles well known in theart. In the case of HLA class I-binding peptides, “minor modifications”refers, in another embodiment, to e.g. insertion, deletion, orsubstitution of one AA, inclusive, or deletion or addition of 1-3 AAoutside of the residues between 2 and 9, inclusive. While the computeralgorithms described herein are useful for predicting the MHC classI-binding potential of peptides, they have 60-80% predictive accuracy;and thus, the peptides should be evaluated empirically before a finaldetermination of MHC class I-binding affinity is made. Thus, peptides ofthe present invention are not limited to peptides predicated by thealgorithms to exhibit strong MHC class I-binding affinity. The types aremodifications that can be made are listed below. Each modificationrepresents a separate embodiment of the present invention.

In another embodiment, a peptide enumerated in the Examples of thepresent invention is further modified by mutating an anchor residue toan MHC class I preferred anchor residue, which can be, in otherembodiments, any of the anchor residues enumerated herein. In anotherembodiment, a peptide of the present invention containing an MHC class Ipreferred anchor residue is further modified by mutating the anchorresidue to a different MHC class I preferred residue for that location.The different preferred residue can be, in other embodiments, any of thepreferred residues enumerated herein.

In another embodiment, the anchor residue that is further modified is inthe 1 position. In another embodiment, the anchor residue is in the 2position. In another embodiment, the anchor residue is in the 3position. In another embodiment, the anchor residue is in the 4position. In another embodiment, the anchor residue is in the 5position. In another embodiment, the anchor residue is in the 6position. In another embodiment, the anchor residue is in the 7position. In another embodiment, the anchor residue is in the 8position. In another embodiment, the anchor residue is in the 9position. In the case of HLA class I-binding peptides, residues otherthan 2 and 9 can serve as secondary anchor residues; therefore, mutatingthem can improve MHC class I binding. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention is a length variant of a peptide enumerated in theExamples. In another embodiment, the length variant is one amino acid(AA) shorter than the peptide from the Examples. In another embodiment,the length variant is two AA shorter than the peptide from the Examples.In another embodiment, the length variant is more than two AA shorterthan the peptide from the Examples. In another embodiment, the shorterpeptide is truncated on the N-terminal end. In another embodiment, theshorter peptide is truncated on the C-terminal end. In anotherembodiment, the truncated peptide is truncated on both the N-terminaland C-terminal ends. Peptides are, in another embodiment, amenable totruncation without changing affinity for HLA molecules, as is well knownin the art.

Each of the above truncated peptides represents a separate embodiment ofthe present invention.

In another embodiment, the length variant is longer than a peptideenumerated in the Examples of the present invention. In anotherembodiment, the longer peptide is extended on the N-terminal end inaccordance with the surrounding WT1 sequence. Peptides are, in anotherembodiment, amenable to extension on the N-terminal end without changingaffinity for HLA molecules, as is well known in the art. Such peptidesare thus equivalents of the peptides enumerated in the Examples. Inanother embodiment, the N-terminal extended peptide is extended by oneresidue. In another embodiment, the N-terminal extended peptide isextended by two residues. In another embodiment, the N-terminal extendedpeptide is extended by three residues. In another embodiment, theN-terminal extended peptide is extended by more than three residues.

In another embodiment, the longer peptide is extended on the C terminalend in accordance with the surrounding WT1 sequence. Peptides are, inanother embodiment, amenable to extension on the C-terminal end withoutchanging affinity for HLA molecules, as is well known in the art. Suchpeptides are thus equivalents of the peptides enumerated in the Examplesof the present invention. In another embodiment, the C-terminal extendedpeptide is extended by one residue. In another embodiment, theC-terminal extended peptide is extended by two residues. In anotherembodiment, the C-terminal extended peptide is extended by threeresidues. In another embodiment, the C-terminal extended peptide isextended by more than three residues.

In another embodiment, the extended peptide is extended on both theN-terminal and C-terminal ends in accordance with the surrounding WT1sequence.

Each of the above extended peptides represents a separate embodiment ofthe present invention.

In another embodiment, a truncated peptide of the present inventionretains the HLA anchor residues (e.g. the HLA class I anchor residues)on the second residue and the C-terminal residue, with a smaller numberof intervening residues (e.g. 5) than a peptide enumerated in theExamples of the present invention. Peptides are, in another embodiment,amenable to such mutation without changing affinity for HLA molecules.In another embodiment, such a truncated peptide is designed by removingone of the intervening residues of one of the above sequences. Inanother embodiment, the HLA anchor residues are retained on the secondand eighth residues. In another embodiment, the HLA anchor residues areretained on the first and eighth residues. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, an extended peptide of the present inventionretains the HLA anchor residues (e.g. the HLA class I anchor residues)on the second residue and the C-terminal residue, with a larger numberof intervening residues (e.g. 7 or 8) than a peptide enumerated in theExamples of the present invention. In another embodiment, such anextended peptide is designed by adding one or more residues between twoof the intervening residues of one of the above sequences. It is wellknown in the art that residues can be removed from or added between theintervening sequences of HLA-binding peptides without changing affinityfor HLA. Such peptides are thus equivalents of the peptides enumeratedin the Examples of the present invention. In another embodiment, the HLAanchor residues are retained on the second and ninth residues. Inanother embodiment, the HLA anchor residues are retained on the firstand eighth residues. In another embodiment, the HLA anchor residues areretained on the two residues separated by six intervening residues. Eachpossibility represents a separate embodiment of the present invention.

“Fragment,” in another embodiment, refers to a peptide of 11 or more AAin length. In another embodiment, a peptide fragment of the presentinvention is 16 or more AA long. In another embodiment, the fragment is12 or more AA long. In another embodiment, the fragment is 13 or moreAA. In another embodiment, the fragment is 14 or more AA. In anotherembodiment, the fragment is 15 or more AA. In another embodiment, thefragment is 17 or more AA. In another embodiment, the fragment is 18 ormore AA. In another embodiment, the fragment is 19 or more AA. Inanother embodiment, the fragment is 22 or more AA. In anotherembodiment, the fragment is 8-12 AA. In another embodiment, the fragmentis about 8-12 AA. In another embodiment, the fragment is 16-19 AA. Inanother embodiment, the fragment is about 16-19 AA. In anotherembodiment, the fragment 10-25 AA. In another embodiment, the fragmentis about 10-25 AA. In another embodiment, the fragment has any otherlength. Each possibility represents a separate embodiment of the presentinvention.

“Fragment of a WT1 protein,” in another embodiment, refers to any of thedefinitions of “fragment” found herein. Each definition represents aseparate embodiment of the present invention.

As provided herein, mesothelioma cells express WT1 protein (Example 7).In addition, mesothelioma cells process and present peptides of thepresent invention or the corresponding native peptides (Example 5).Moreover, the presentation is robust enough to elicit anti-WT1 specificimmune responses (Example 5). Thus, mesothelioma cells can be targetedby anti-WT1 immune therapy.

In another embodiment, a peptide of the present invention is homologousto a peptide enumerated in the Examples. The terms “homology,”“homologous,” etc, when in reference to any protein or peptide, refer,in another embodiment, to a percentage of amino acid residues in thecandidate sequence that are identical with the residues of acorresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Methods and computer programs for the alignment arewell known in the art.

In another embodiment, the term “homology,” when in reference to anynucleic acid sequence similarly indicates a percentage of nucleotides ina candidate sequence that are identical with the nucleotides of acorresponding native nucleic acid sequence.

Homology is, in another embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. In otherembodiments, computer algorithm analysis of nucleic acid sequencehomology includes the utilization of any number of software packagesavailable, such as, for example, the BLAST, DOMAIN, BEAUTY (BLASTEnhanced Alignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-38 of greater than 70%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 1-38 of greater than 72%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 1-38 of greater than 75%. Inanother embodiment, “homology” refers to identity to a sequence selectedfrom SEQ ID No: 1-38 of greater than 78%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 1-38 of greater than80%. In another embodiment, “homology” refers to identity to one of SEQID No: 1-38 of greater than 82%. In another embodiment, “homology”refers to identity to a sequence selected from SEQ ID No: 1-38 ofgreater than 83%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 1-38 of greater than 85%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 1-38 of greater than87%. In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-38 of greater than 88%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-38 ofgreater than 90%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 1-38 of greater than 92%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No:1-38 of greater than 93%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 1-38 of greater than 95%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 1-38 of greater than 96%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 1-38 of greater than 97%. Inanother embodiment, “homology” refers to identity to one of SEQ ID No:1-38 of greater than 98%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 1-38 of greater than 99%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-38 of100%. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, homology is determined via determination ofcandidate sequence hybridization, methods of which are well described inthe art (See, for example, “Nucleic Acid Hybridization” Hames, B. D.,and Higgins S. J., Eds. (1985); Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y). In anotherembodiments, methods of hybridization are carried out under moderate tostringent conditions, to the complement of a DNA encoding a nativecaspase peptide. Hybridization conditions being, for example, overnightincubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA.

Each of the above homologues and variants of peptides enumerated in theExamples represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising a peptide of this invention. In another embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inanother embodiment, the composition further comprises an adjuvant. Inanother embodiment, the composition comprises 2 or more peptides of thepresent invention. In another embodiment, the composition furthercomprises any of the additives, compounds, or excipients set forthhereinbelow. In another embodiment, the adjuvant is KLH, QS21, Freund'scomplete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide,BCG or alum. In other embodiments, the carrier is any carrier enumeratedherein. In other embodiments, the adjuvant is any adjuvant enumeratedherein. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, this invention provides a vaccine comprising apeptide of this invention. In another embodiment, this inventionprovides a vaccine comprising an antigen-presenting cell (APC) and apeptide of this invention. In another embodiment, the vaccine furthercomprises a carrier. In another embodiment, the vaccine furthercomprises an adjuvant. In another embodiment, the vaccine furthercomprises an APC. In another embodiment, the vaccine further comprises acombination of more than 1 of an antigen, carrier, and/or APC. Inanother embodiment, the vaccine is a cell-based composition. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the term “vaccine” refers to a material orcomposition that, when introduced into a subject, provides aprophylactic or therapeutic response for a particular disease,condition, or symptom of same. In another embodiment, this inventioncomprises peptide-based vaccines, wherein the peptide comprises anyembodiment listed herein, including immunomodulating compounds such ascytokines, adjuvants, etc.

In another embodiment, a vaccine of methods and compositions of thepresent invention further comprises an adjuvant. In another embodiment,the adjuvant is Montanide ISA 51. Montanide ISA 51 contains a naturalmetabolizable oil and a refined emulsifier. In another embodiment, theadjuvant is GM-CSF. Recombinant GM-CSF is a human protein grown, inanother embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotesclonal expansion and differentiation of hematopoietic progenitor cells,APC, and dendritic cells and T cells.

In another embodiment, the adjuvant is a cytokine. In anotherembodiment, the adjuvant is a growth factor. In another embodiment, theadjuvant is a cell population. In another embodiment, the adjuvant isQS21. In another embodiment, the adjuvant is Freund's incompleteadjuvant. In another embodiment, the adjuvant is aluminum phosphate. Inanother embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is BCG. In another embodiment, the adjuvant isalum. In another embodiment, the adjuvant is an interleukin. In anotherembodiment, the adjuvant is a chemokine. In another embodiment, theadjuvant is any other type of adjuvant known in the art. In anotherembodiment, the WT1 vaccine comprises two the above adjuvants. Inanother embodiment, the WT1 vaccine comprises more than two the aboveadjuvants. Each possibility represents a separate embodiment of thepresent invention.

In other embodiments, a vaccine or composition of the present inventioncan comprise any of the embodiments of WT1 peptides of the presentinvention and combinations thereof. Each possibility represents aseparate embodiment of the present invention.

It is to be understood that any embodiments described herein, regardingpeptides, vaccines and compositions of this invention can be employed inany of the methods of this invention. Each combination of peptide,vaccine, or composition with a method represents an embodiment thereof.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a WT1 vaccine of the present invention,thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method oftreating a subject with an MDS, the method comprising administering tothe subject a WT1 vaccine of the present invention, thereby treating asubject with an MDS.

In another embodiment, the present invention provides a method ofsuppressing or halting the progression of a WT1-expressing cancer in asubject, the method comprising administering to the subject a WT1vaccine of the present invention, thereby suppressing or halting theprogression of a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer in a subject, themethod comprising administering to the subject a WT1 vaccine of thepresent invention, thereby reducing the incidence of a WT1-expressingcancer in a subject.

In another embodiment, the present invention provides a method ofreducing the incidence of an AML in a subject, the method comprisingadministering to the subject a WT1 vaccine of the present invention,thereby reducing the incidence of an AML.

In another embodiment, the present invention provides a method ofreducing the incidence of relapse of a WT1-expressing cancer in asubject, the method comprising administering to the subject a WT1vaccine of the present invention, thereby reducing the incidence ofrelapse of a WT1-expressing cancer in a subject.

In another embodiment, the present invention provides a method ofreducing the incidence of relapse of an AML in a subject, the methodcomprising administering to the subject a WT1 vaccine of the presentinvention, thereby reducing the incidence of relapse of an AML in asubject.

In another embodiment, the present invention provides a method ofbreaking a T cell tolerance of a subject to a WT1-expressing cancer, themethod comprising administering to the subject a WT1 vaccine of thepresent invention, thereby breaking a T cell tolerance to aWT1-expressing cancer.

In another embodiment, the present invention provides a method oftreating a subject having a WT1-expressing cancer, comprising (a)inducing in a donor formation and proliferation of human cytotoxic Tlymphocytes (CTL) that recognize a malignant cell of the cancer by amethod of the present invention; and (b) infusing the human CTL into thesubject, thereby treating a subject having a cancer.

In another embodiment, the present invention provides a method oftreating a subject having a WT1-expressing cancer, comprising (a)inducing ex vivo formation and proliferation of human CTL that recognizea malignant cell of the cancer by a method of the present invention,wherein the human immune cells are obtained from a donor, and (b)infusing the human CTL into the subject, thereby treating a subjecthaving a cancer.

Methods for ex vivo immunotherapy are well known in the art and aredescribed, for example, in Davis I D et al (Blood dendritic cellsgenerated with Flt3 ligand and CD40 ligand prime CD8+ T cellsefficiently in cancer patients. J Immunother. 2006 September-October;29(5):499-511) and Mitchell M S et al (The cytotoxic T cell response topeptide analogs of the HLA-A*0201-restricted MUC1 signal sequenceepitope, M1.2. Cancer Immunol Immunother. 2006 Jul. 28). Each methodrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofinducing the formation and proliferation of CTL specific for cells of aWT1-expressing cancer, the method comprising contacting a lymphocytepopulation with a vaccine of the present invention. In anotherembodiment, the vaccine is an APC associated with a peptide of thepresent invention. In another embodiment, the vaccine is an APCassociated with a mixture of peptides of the present invention. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, this invention provides a method of generating aheteroclitic immune response in a subject, wherein the heterocliticimmune response is directed against a WT1-expressing cancer, the methodcomprising administering to the subject a vaccine of the presentinvention, thereby generating a heteroclitic immune response.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT1 protein; or (b) a fragment of a WTprotein, thereby inducing an anti-mesothelioma immune response in asubject. In another embodiment, the mesothelioma is a malignantmesothelioma. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising a nucleotide molecule encoding (a) a WT1 protein;or (b) a fragment of a WT1 protein, thereby inducing ananti-mesothelioma immune response in a subject. In another embodiment,the mesothelioma is a malignant mesothelioma. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising(a) a WT1 protein; or (b) a fragment of a WT protein, thereby treating asubject with a mesothelioma. In another embodiment, the mesothelioma isa malignant mesothelioma. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising anucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of aWT1 protein, thereby treating a subject with a mesothelioma. In anotherembodiment, the mesothelioma is a malignant mesothelioma. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising (a) a WT1 protein; or (b) a fragmentof a WT protein, thereby reducing an incidence of a mesothelioma, or itsrelapse, in a subject. In another embodiment, the mesothelioma is amalignant mesothelioma. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising a nucleotide molecule encoding (a) aWT1 protein; or (b) a fragment of a WT1 protein, thereby reducing anincidence of a mesothelioma, or its relapse, in a subject. In anotherembodiment, the mesothelioma is a malignant mesothelioma. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a target cell of an immune response elicited by amethod of the present invention presents the WT1 peptide of the presentinvention, or a corresponding WT1 fragment, on an HLA molecule. Inanother embodiment, the HLA molecule is an HLA class I molecule. Inother embodiments, the HLA molecule is any HLA class I subtype or HLAclass I molecule known in the art. In another embodiment, the immuneresponse against the WT1 peptide or fragment is a heteroclitic immuneresponse. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the WT1-expressing cancer is an acute myelogenousleukemia (AML). In another embodiment, the WT1-expressing cancer isassociated with a myelodysplastic syndrome (MDS). In another embodiment,the WT1-expressing cancer is an MDS. In another embodiment, theWT1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT1-expressing cancer is a leukemia. In anotherembodiment, the WT1-expressing cancer is a hematological cancer. Inanother embodiment, the WT1-expressing cancer is a lymphoma. In anotherembodiment, the WT1-expressing cancer is a desmoplastic small round celltumor. In another embodiment, the WT1-expressing cancer is amesothelioma. In another embodiment, the WT1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT1-expressing canceris a gastric cancer. In another embodiment, the WT1-expressing cancer isa colon cancer. In another embodiment, the WT1-expressing cancer is alung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is agerm cell tumor. In another embodiment, the WT1-expressing cancer is anovarian cancer. In another embodiment, the WT1-expressing cancer is auterine cancer. In another embodiment, the WT1-expressing cancer is athyroid cancer. In another embodiment, the WT1-expressing cancer is ahepatocellular carcinoma. In another embodiment, the WT1-expressingcancer is a thyroid cancer. In another embodiment, the WT1-expressingcancer is a liver cancer. In another embodiment, the WT1-expressingcancer is a renal cancer. In another embodiment, the WT1-expressingcancer is a kaposi's sarcoma. In another embodiment, the WT1-expressingcancer is a sarcoma. In another embodiment, the WT1-expressing cancer isany other carcinoma or sarcoma.

In another embodiment, the WT1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, a cancer or tumor treated by a method of thepresent invention is suspected to express WT1. In another embodiment,WT1 expression has not been verified by testing of the actual tumorsample. In another embodiment, the cancer or tumor is of a type known toexpress WT1 in many cases. In another embodiment, the type expresses WT1in the majority of cases.

Each type of WT1-expressing cancer or tumor, and cancer or tumorsuspected to express WT1, represents a separate embodiment of thepresent invention.

Any embodiments enumerated herein, regarding peptides, vaccines andcompositions of this invention can be employed in any of the methods ofthis invention, and each represents an embodiment thereof.

In another embodiment, multiple peptides of this invention are used tostimulate an immune response in methods of the present invention.

As provided herein, peptides of the present invention elicitantigen-specific CD8⁺ T cell responses (Examples 1-2) and CD4⁺ T cellresponses (Examples 3-4). CD4⁺ T cells recognize peptides bound to theHLA class II molecule on APC. In another embodiment, antigen-specificCD4⁺ T cell responses assist in induction and maintenance of CD8⁺cytotoxic T cell (CTL) responses. In another embodiment, activated CD4⁺cells enhance immunity by licensing dendritic cells, thereby sustainingthe activation and survival of the cytotoxic T cells. In anotherembodiment, activated CD4⁺ T cells induce tumor cell death by directcontact with the tumor cell or by activation of the apoptosis pathway.Mesothelioma tumor cells, for example, are able to process and presentantigens in the context of HLA class I and class II molecules.

In another embodiment, vaccines of the present invention have theadvantage of activating or eliciting both CD4⁺ and CD8⁺ T cells thatrecognize WT1 antigens. In another embodiment, activation or elicitingboth CD4⁺ and CD8⁺ T cells provides a synergistic anti-WT1 immuneresponse, relative to activation of either population alone.

The methods disclosed herein will be understood by those in the art toenable design of other WT1-derived peptides. The methods further enabledesign of peptides binding to other HLA molecules. The methods furtherenable design of vaccines combining WT1-derived peptides of the presentinvention. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, vaccines of the present invention have theadvantage of activating or eliciting WT1-specific CD4⁺ T cellscontaining a variety of different HLA class II alleles. In anotherembodiment, the vaccines have the advantage of activating or elicitingWT1-specific CD4⁺ T cells in a substantial proportion of the population(e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%,90%, 95%, or greater than 95%). In another embodiment, the vaccinesactivate or elicit WT1-specific CD4⁺ T cells in a substantial proportionof a particular population (e.g. American Caucasians). Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, methods of the present invention provide for animprovement in an immune response that has already been mounted by asubject. In another embodiment, methods of the present inventioncomprise administering the peptide, composition, or vaccine 2 or moretimes. In another embodiment, the peptides are varied in theircomposition, concentration, or a combination thereof. In anotherembodiment, the peptides provide for the initiation of an immuneresponse against an antigen of interest in a subject who has not yetinitiated an immune response against the antigen. In another embodiment,the CTL that are induced proliferate in response to presentation of thepeptide on the APC or cancer cell. In other embodiments, reference tomodulation of the immune response involves, either or both the humoraland cell-mediated arms of the immune system, which is accompanied by thepresence of Th2 and Th1 T helper cells, respectively, or in anotherembodiment, each arm individually.

In other embodiments, the methods affecting the growth of a tumor resultin (1) the direct inhibition of tumor cell division, or (2) immune cellmediated tumor cell lysis, or both, which leads to a suppression in thenet expansion of tumor cells.

Inhibition of tumor growth by either of these two mechanisms can bereadily determined by one of ordinary skill in the art based upon anumber of well known methods. In another embodiment, tumor inhibition isdetermined by measuring the actual tumor size over a period of time. Inanother embodiment, tumor inhibition can be determined by estimating thesize of a tumor (over a period of time) utilizing methods well known tothose of skill in the art. More specifically, a variety of radiologicimaging methods (e.g., single photon and positron emission computerizedtomography; see generally, “Nuclear Medicine in Clinical Oncology,”Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized toestimate tumor size. Such methods can also utilize a variety of imagingagents, including for example, conventional imaging agents (e.g.,Gallium-67 citrate), as well as specialized reagents for metaboliteimaging, receptor imaging, or immunologic imaging (e.g., radiolabeledmonoclonal antibody specific tumor markers). In addition,non-radioactive methods such as ultrasound (see, “UltrasonicDifferential Diagnosis of Tumors”, Kossoff and Fukuda, (eds.),Igaku-Shoin, New York, 1984), can also be utilized to estimate the sizeof a tumor.

In addition to the in vivo methods for determining tumor inhibitiondiscussed above, a variety of in vitro methods can be utilized in orderto predict in vivo tumor inhibition. Representative examples includelymphocyte mediated anti-tumor cytolytic activity determined forexample, by a ⁵¹Cr release assay (Examples), tumor dependent lymphocyteproliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991),in vitro generation of tumor specific antibodies (Herlyn, et al., J.Immunol. Meth. 73:157-167, 1984), cell (e.g., CTL, helper T-cell) orhumoral (e.g., antibody) mediated inhibition of cell growth in vitro(Gazit, et al., Cancer Immunol Immunother 35:135-144, 1992), and, forany of these assays, determination of cell precursor frequency (Vose,Int. J. Cancer 30:135-142 (1982), and others.

In another embodiment, methods of suppressing tumor growth indicate agrowth state that is curtailed compared to growth without contact with,or exposure to a peptide of this invention. Tumor cell growth can beassessed by any means known in the art, including, but not limited to,measuring tumor size, determining whether tumor cells are proliferatingusing a ³H-thymidine incorporation assay, or counting tumor cells.“Suppressing” tumor cell growth refers, in other embodiments, toslowing, delaying, or stopping tumor growth, or to tumor shrinkage. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, WT1 expression is measured. In another embodiment, WT1transcript expression is measured. In another embodiment, WT1 proteinlevels in the tumor are measured. Each possibility represents a separateembodiment of the present invention.

Methods of determining the presence and magnitude of an immune responseare well known in the art. In another embodiment, lymphocyteproliferation assays, wherein T cell uptake of a radioactive substance,e.g. ³H-thymidine is measured as a function of cell proliferation. Inother embodiments, detection of T cell proliferation is accomplished bymeasuring increases in interleukin-2 (IL-2) production, Ca²⁺ flux, ordye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, CTL stimulation is determined by means known tothose skilled in the art, including, detection of cell proliferation,cytokine production and others. Analysis of the types and quantities ofcytokines secreted by T cells upon contacting ligand-pulsed targets canbe a measure of functional activity. Cytokines can be measured by ELISAor ELISPOT assays to determine the rate and total amount of cytokineproduction. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160:181;Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).

In another embodiment, CTL activity is determined by ⁵¹Cr-release lysisassay. Lysis of peptide-pulsed ⁵¹Cr-labeled targets by antigen-specificT cells can be compared for target cells pulsed with control peptide. Inanother embodiment, T cells are stimulated with a peptide of thisinvention, and lysis of target cells expressing the native peptide inthe context of MHC can be determined. The kinetics of lysis as well asoverall target lysis at a fixed timepoint (e.g., 4 hours) are used, inanother embodiment, to evaluate ligand performance. (Ware C. F. et al.(1983) J Immunol 131: 1312).

Methods of determining affinity of a peptide for an HLA molecule arewell known in the art. In another embodiment, affinity is determined byTAP stabilization assays (Examples).

In another embodiment, affinity is determined by competitionradioimmunoassay. In another embodiment, the following protocol isutilized: Target cells are washed two times in PBS with 1% bovine serumalbumin (BSA; Fisher Chemicals, Fairlawn, N.J.). Cells are resuspendedat 10⁷/ml on ice, and the native cell surface bound peptides arestripped for 2 minutes at 0° C. using citrate-phosphate buffer in thepresence of 3 mg/ml beta₂ microglobulin. The pellet is resuspended at5×10⁶ cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta₂microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots areincubated in the presence or absence of HLA-specific peptides for 10 minat 20° C., then with ¹²⁵I-labeled peptide for 30 min at 20° C. Totalbound ¹²⁵I is determined after two washes with PBS/2% BSA and one washwith PBS. Relative affinities are determined by comparison of escalatingconcentrations of the test peptide versus a known binding peptide.

In another embodiment, a specificity analysis of the binding of peptideto HLA on surface of live cells (e.g. SKLY-16 cells) is conducted toconfirm that the binding is to the appropriate HLA molecule and tocharacterize its restriction. This includes, in another embodiment,competition with excess unlabeled peptides known to bind to the same ordisparate HLA molecules and use of target cells which express the sameor disparate HLA types. This assay is performed, in another embodiment,on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia celllines and EBV-transformed T-cell lines of specific HLA types. Therelative avidity of the peptides found to bind MHC molecules on thespecific cells are assayed by competition assays as described aboveagainst ¹²⁵I-labeled peptides of known high affinity for the relevantHLA molecule, e.g., tyrosinase or HBV peptide sequence.

In another embodiment, an HLA class II-binding peptide of methods andcompositions of the present invention is longer than the minimum lengthfor binding to an HLA class II molecule, which is, in anotherembodiment, about 12 AA. In another embodiment, increasing the length ofthe HLA class II-binding peptide enables binding to more than one HLAclass II molecule. In another embodiment, increasing the length enablesbinding to an HLA class II molecule whose binding motif is not known. Inanother embodiment, increasing the length enables binding to an HLAclass I molecule. In another embodiment, the binding motif of the HLAclass I molecule is known. In another embodiment, the binding motif ofthe HLA class I molecule is not known. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the peptides utilized in methods and compositionsof the present invention comprise a non-classical amino acid such as:1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991)J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) TetrahedronLett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid(Landis (1989) Ph.D. Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1984) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); andHIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep.Protein Res. 42(1):68-77) and ((1992) Acta. Crst., Crystal Struc. Comm.48(IV):1239-124).

In another embodiment, a peptide of this invention comprises an AAanalog or peptidomimetic, which, in other embodiments, induces or favorsspecific secondary structures. Such peptides comprise, in otherembodiments, the following: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a ß-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838);ß-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett.29:5081-5082); ß-turn inducing analogs (Kemp et al. (1988) TetrahedronLeft. 29:5057-5060); alpha-helix inducing analogs (Kemp et al. (1988)Tetrahedron Left. 29:4935-4938); gamma-turn inducing analogs (Kemp etal. (1989) J. Org. Chem. 54:109:115); analogs provided by the followingreferences: Nagai and Sato (1985) Tetrahedron Left. 26:647-650; andDiMaio et al. (1989) J. Chem. Soc. Perkin Trans. p. 1687; a Gly-Ala turnanalog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bondisostere (Jones et al. (1988) Tetrahedron Left. 29(31):3853-3856);tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC(Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); andanalogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 andGarvey et al. (1990) J. Org. Chem. 55(3):936-940. Conformationallyrestricted mimetics of beta turns and beta bulges, and peptidescontaining them, are described in U.S. Pat. No. 5,440,013, issued Aug.8, 1995 to Kahn.

In other embodiments, a peptide of this invention is conjugated to oneof various other molecules, as described hereinbelow, which can be viacovalent or non-covalent linkage (complexed), the nature of whichvaries, in another embodiment, depending on the particular purpose. Inanother embodiment, the peptide is covalently or non-covalentlycomplexed to a macromolecular carrier, (e.g. an immunogenic carrier),including, but not limited to, natural and synthetic polymers, proteins,polysaccharides, polypeptides (amino acids), polyvinyl alcohol,polyvinyl pyrrolidone, and lipids. In another embodiment, a peptide ofthis invention is linked to a substrate. In another embodiment, thepeptide is conjugated to a fatty acid, for introduction into a liposome(U.S. Pat. No. 5,837,249). In another embodiment, a peptide of theinvention is complexed covalently or non-covalently with a solidsupport, a variety of which are known in the art. In another embodiment,linkage of the peptide to the carrier, substrate, fatty acid, or solidsupport serves to increase an elicited an immune response.

In other embodiments, the carrier is thyroglobulin, an albumin (e.g.human serum albumin), tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), an influenza protein, hepatitis B virus coreprotein, keyhole limpet hemocyanin, an albumin, or another carrierprotein or carrier peptide; hepatitis B virus recombinant vaccine, or anAPC. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the term “amino acid” (AA) refers to a naturalor, in another embodiment, an unnatural or synthetic AA, and caninclude, in other embodiments, glycine, D- or L optical isomers, AAanalogs, peptidomimetics, or combinations thereof.

In another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or“tumor,” are used interchangeably and refer to cells that have undergonea malignant transformation that makes them pathological to the hostorganism. Primary cancer cells (that is, cells obtained from near thesite of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.In another embodiment, a tumor is detectable on the basis of tumor mass;e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI),X-ray, ultrasound or palpation, and in another embodiment, is identifiedby biochemical or immunologic findings, the latter which is used toidentify cancerous cells, as well, in other embodiments.

Methods for synthesizing peptides are well known in the art. In anotherembodiment, the peptides of this invention are synthesized using anappropriate solid-state synthetic procedure (see for example, Stewardand Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco,Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23:451). The activity of these peptides is tested, in other embodiments,using assays as described herein.

In another embodiment, the peptides of this invention are purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for protein purification.In another embodiment, immuno-affinity chromatography is used, wherebyan epitope is isolated by binding it to an affinity column comprisingantibodies that were raised against that peptide, or a related peptideof the invention, and were affixed to a stationary support.

In another embodiment, affinity tags such as hexa-His (Invitrogen),Maltose binding domain (New England Biolabs), influenza coat sequence(Kolodziej et al. (1991) Meth. Enzymol. 194:508-509),glutathione-S-transferase, or others, are attached to the peptides ofthis invention to allow easy purification by passage over an appropriateaffinity column. Isolated peptides can also be physically characterized,in other embodiments, using such techniques as proteolysis, nuclearmagnetic resonance, and x-ray crystallography.

In another embodiment, the peptides of this invention are produced by invitro translation, through known techniques, as will be evident to oneskilled in the art. In another embodiment, the peptides aredifferentially modified during or after translation, e.g., byphosphorylation, glycosylation, cross-linking, acylation, proteolyticcleavage, linkage to an antibody molecule, membrane molecule or otherligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).

In another embodiment, the peptides of this invention further comprise adetectable label, which in another embodiment, is fluorescent, or inanother embodiment, luminescent, or in another embodiment, radioactive,or in another embodiment, electron dense. In other embodiments, thedetectable label comprises, for example, green fluorescent protein(GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase(SEAP), beta-galactosidase, luciferase, ³²P, ¹²⁵I, ³H and ¹⁴C,fluorescein and its derivatives, rhodamine and its derivatives, dansyland umbelliferone, luciferin or any number of other such labels known toone skilled in the art. The particular label used will depend upon thetype of immunoassay used.

In another embodiment, a peptide of this invention is linked to asubstrate, which, in another embodiment, serves as a carrier. In anotherembodiment, linkage of the peptide to a substrate serves to increase anelicited an immune response.

In another embodiment, peptides of this invention are linked to othermolecules, as described herein, using conventional cross-linking agentssuch as carbodimides. Examples of carbodimides are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

In other embodiments, the cross-linking agents comprise cyanogenbromide, glutaraldehyde and succinic anhydride. In general, any of anumber of homo-bifunctional agents including a homo-bifunctionalaldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester,a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound can be used. Also envisioned,in other embodiments, are hetero-bifunctional compounds, for example,compounds having an amine-reactive and a sulfhydryl-reactive group,compounds with an amine-reactive and a photoreactive group and compoundswith a carbonyl-reactive and a sulfhydryl-reactive group.

In other embodiments, the homo-bifunctional cross-linking agents includethe bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartarate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio)propionamido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether, the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as a1a′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively.

In other embodiments, hetero-bifunctional cross-linking agents used tolink the peptides to other molecules, as described herein, include, butare not limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(.gamma.-maleimidobutyryloxy)succinimide ester), MPBH(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

In another embodiment, the peptides of the invention are formulated asnon-covalent attachment of monomers through ionic, adsorptive, orbiospecific interactions. Complexes of peptides with highly positivelyor negatively charged molecules can be accomplished, in anotherembodiment, through salt bridge formation under low ionic strengthenvironments, such as in deionized water. Large complexes can becreated, in another embodiment, using charged polymers such aspoly-(L-glutamic acid) or poly-(L-lysine), which contain numerousnegative and positive charges, respectively. In another embodiment,peptides are adsorbed to surfaces such as microparticle latex beads orto other hydrophobic polymers, forming non-covalently associatedpeptide-superantigen complexes effectively mimicking cross-linked orchemically polymerized protein, in other embodiments. In anotherembodiment, peptides are non-covalently linked through the use ofbiospecific interactions between other molecules. For instance,utilization of the strong affinity of biotin for proteins such as avidinor streptavidin or their derivatives could be used to form peptidecomplexes. The peptides, according to this aspect, and in anotherembodiment, can be modified to possess biotin groups using commonbiotinylation reagents such as the N-hydroxysuccinimidyl ester ofD-biotin (NHS-biotin), which reacts with available amine groups.

In another embodiment, a peptide of the present invention is linked to acarrier. In another embodiment, the carrier is KLH. In otherembodiments, the carrier is any other carrier known in the art,including, for example, thyroglobulin, albumins such as human serumalbumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamicacid), influenza, hepatitis B virus core protein, hepatitis B virusrecombinant vaccine and the like. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the peptides of this invention are conjugated toa lipid, such as P3 CSS. In another embodiment, the peptides of thisinvention are conjugated to a bead.

In another embodiment, the compositions of this invention furthercomprise immunomodulating compounds. In other embodiments, theimmunomodulating compound is a cytokine, chemokine, or complementcomponent that enhances expression of immune system accessory oradhesion molecules, their receptors, or combinations thereof. In someembodiments, the immunomodulating compound include interleukins, forexample interleukins 1 to 15, interferons alpha, beta or gamma, tumournecrosis factor, granulocyte-macrophage colony stimulating factor(GM-CSF), macrophage colony stimulating factor (M-CSF), granulocytecolony stimulating factor (G-CSF), chemokines such as neutrophilactivating protein (NAP), macrophage chemoattractant and activatingfactor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a andMIP-1b, complement components, or combinations thereof. In otherembodiments, the immunomodulating compound stimulate expression, orenhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L,B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combinationthereof.

In another embodiment, the immunomodulatory compound induces or enhancesexpression of co-stimulatory molecules that participate in the immuneresponse, which include, in some embodiments, CD40 or its ligand, CD28,CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatorycompound induces or enhances expression of a heat stable antigen (HSA)(Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitinsulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al.(1993) Cell 74:257-268), or an intracellular adhesion molecule 1(ICAM-1) (Van R. H. (1992) Cell 71:1065-1068), which assists, in anotherembodiment, co-stimulation by interacting with their cognate ligands onthe T cells.

In another embodiment, the composition comprises a solvent, includingwater, dispersion media, cell culture media, isotonic agents and thelike. In another embodiment, the solvent is an aqueous isotonic bufferedsolution with a pH of around 7.0. In another embodiment, the compositioncomprises a diluent such as water, phosphate buffered saline, or saline.In another embodiment, the composition comprises a solvent, which isnon-aqueous, such as propyl ethylene glycol, polyethylene glycol andvegetable oils.

In another embodiment, the composition is formulated for administrationby any of the many techniques known to those of skill in the art. Forexample, this invention provides for administration of thepharmaceutical composition parenterally, intravenously, subcutaneously,intradermally, intramucosally, topically, orally, or by inhalation.

In another embodiment, the vaccine comprising a peptide of thisinvention further comprises a cell population, which, in anotherembodiment, comprises lymphocytes, monocytes, macrophages, dendriticcells, endothelial cells, stem cells or combinations thereof, which, inanother embodiment are autologous, syngeneic or allogeneic, with respectto each other. In another embodiment, the cell population comprises apeptide of the present invention. In another embodiment, the cellpopulation takes up the peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the cell populations of this invention areobtained from in vivo sources, such as, for example, peripheral blood,leukopheresis blood product, apheresis blood product, peripheral lymphnodes, gut associated lymphoid tissue, spleen, thymus, cord blood,mesenteric lymph nodes, liver, sites of immunologic lesions, e.g.synovial fluid, pancreas, cerebrospinal fluid, tumor samples,granulomatous tissue, or any other source where such cells can beobtained. In another embodiment, the cell populations are obtained fromhuman sources, which are, in other embodiments, from human fetal,neonatal, child, or adult sources. In another embodiment, the cellpopulations of this invention are obtained from animal sources, such as,for example, porcine or simian, or any other animal of interest. Inanother embodiment, the cell populations of this invention are obtainedfrom subjects that are normal, or in another embodiment, diseased, or inanother embodiment, susceptible to a disease of interest.

In another embodiment, the cell populations of this invention areseparated via affinity-based separation methods. Techniques for affinityseparation include, in other embodiments, magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or use in conjunction with amonoclonal antibody, for example, complement and cytotoxins, and“panning” with an antibody attached to a solid matrix, such as a plate,or any other convenient technique. In other embodiment, separationtechniques include the use of fluorescence activated cell sorters, whichcan have varying degrees of sophistication, such as multiple colorchannels, low angle and obtuse light scattering detecting channels,impedance channels, etc. In other embodiments, any technique thatenables separation of the cell populations of this invention can beemployed, and is to be considered as part of this invention.

In another embodiment, the dendritic cells are from the diversepopulation of morphologically similar cell types found in a variety oflymphoid and non-lymphoid tissues, qualified as such (Steinman (1991)Ann. Rev. Immunol. 9:271-296). In another embodiment, the dendriticcells used in this invention are isolated from bone marrow, or inanother embodiment, derived from bone marrow progenitor cells, or, inanother embodiment, from isolated from/derived from peripheral blood, orin another embodiment, derived from, or are a cell line.

In another embodiment, the cell populations described herein areisolated from the white blood cell fraction of a mammal, such as amurine, simian or a human (See, e.g., WO 96/23060). The white blood cellfraction can be, in another embodiment, isolated from the peripheralblood of the mammal.

Methods of isolating dendritic cells are well known in the art. Inanother embodiment, the DC are isolated via a method which includes thefollowing steps: (a) providing a white blood cell fraction obtained froma mammalian source by methods known in the art such as leukophoresis;(b) separating the white blood cell fraction of step (a) into four ormore subfractions by countercurrent centrifugal elutriation; (c)stimulating conversion of monocytes in one or more fractions from step(b) to dendritic cells by contacting the cells with calcium ionophore,GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendriticcell-enriched fraction from step (c); and (e) collecting the enrichedfraction of step (d), preferably at about 4° C.

In another embodiment, the dendritic cell-enriched fraction isidentified by fluorescence-activated cell sorting, which identifies atleast one of the following markers: HLA-DR, HLA-DQ, or B7.2, and thesimultaneous absence of the following markers: CD3, CD14, CD16, 56, 57,and CD19, 20.

In another embodiment, the cell population comprises lymphocytes, whichare, in another embodiment, T cells, or in another embodiment, B cells.The T cells are, in other embodiments, characterized as NK cells, helperT cells, cytotoxic T lymphocytes (CTL), TILs, naïve T cells, orcombinations thereof. It is to be understood that T cells which areprimary, or cell lines, clones, etc. are to be considered as part ofthis invention. In another embodiment, the T cells are CTL, or CTLlines, CTL clones, or CTLs isolated from tumor, inflammatory, or otherinfiltrates.

In another embodiment, hematopoietic stem or early progenitor cellscomprise the cell populations used in this invention. In anotherembodiment, such populations are isolated or derived, by leukophoresis.In another embodiment, the leukopheresis follows cytokineadministration, from bone marrow, peripheral blood (PB) or neonatalumbilical cord blood. In another embodiment, the stem or progenitorcells are characterized by their surface expression of the surfaceantigen marker known as CD34⁺, and exclusion of expression of thesurface lineage antigen markers, Lin−.

In another embodiment, the subject is administered a peptide,composition or vaccine of this invention, in conjunction with bonemarrow cells. In another embodiment, the administration together withbone marrow cells embodiment follows previous irradiation of thesubject, as part of the course of therapy, in order to suppress, inhibitor treat cancer in the subject.

In another embodiment, the phrase “contacting a cell” or “contacting apopulation” refers to a method of exposure, which can be, in otherembodiments, direct or indirect. In another embodiment, such contactcomprises direct injection of the cell through any means well known inthe art, such as microinjection. It is also envisaged, in anotherembodiment, that supply to the cell is indirect, such as via provisionin a culture medium that surrounds the cell, or administration to asubject, via any route well known in the art, and as described herein.

In another embodiment, CTL generation of methods of the presentinvention is accomplished in vivo, and is effected by introducing into asubject an antigen presenting cell contacted in vitro with a peptide ofthis invention (See for example Paglia et al. (1996) J. Exp. Med.183:317-322).

In another embodiment, the peptides of methods and compositions of thepresent invention are delivered to APC. In another embodiment, thepeptide-pulsed APC are administered to a subject to elicit and immuneresponse or treat or inhibit growth or recurrence of a tumor. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the peptides are delivered to APC in the form ofcDNA encoding the peptides. In another embodiment, the term“antigen-presenting cells” (APC) refers to dendritic cells (DC),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules, which effectively allow forT cell recognition of the presented peptide. In another embodiment, theAPC is a cancer cell. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the CTL are contacted with 2 or more APCpopulations. In another embodiment, the 2 or more APC populationspresent different peptides. Each possibility represents a separateembodiment of the present invention.

In another embodiment, techniques that lead to the expression of antigenin the cytosol of APC (e.g. DC) are used to deliver the peptides to theAPC. Methods for expressing antigens on APC are well known in the art.In another embodiment, the techniques include (1) the introduction intothe APC of naked DNA encoding a peptide of this invention, (2) infectionof APC with recombinant vectors expressing a peptide of this invention,and (3) introduction of a peptide of this invention into the cytosol ofan APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med.184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair et al.(1992) J. Exp. Med. 175:609-612).

In another embodiment, foster APC such as those derived from the humancell line 174xCEM.T2, referred to as T2, which contains a mutation inits antigen processing pathway that restricts the association ofendogenous peptides with cell surface MHC class I molecules (Zweerink etal. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein.

In another embodiment, as described herein, the subject is exposed to apeptide, or a composition/cell population comprising a peptide of thisinvention, which differs from the native protein expressed, whereinsubsequently a host immune cross-reactive with the nativeprotein/antigen develops.

In another embodiment, the subject, as referred to in any of the methodsor embodiments of this invention is a human. In other embodiments, thesubject is a mammal, which can be a mouse, rat, rabbit, hamster, guineapig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, peptides, vaccines, and compositions of thisinvention stimulate an immune response that results in tumor cell lysis.

In another embodiment, any of the methods described herein is used toelicit CTL, which are elicited in vitro. In another embodiment, the CTLare elicited ex-vivo. In another embodiment, the CTL are elicited invitro. The resulting CTL, are, in another embodiment, administered tothe subject, thereby treating the condition associated with the peptide,an expression product comprising the peptide, or a homologue thereof.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the method entails introduction of the geneticsequence that encodes the peptides of this invention. In anotherembodiment, the method comprises administering to the subject a vectorcomprising a nucleotide sequence, which encodes a peptide of the presentinvention (Tindle, R. W. et al. Virology (1994) 200:54). In anotherembodiment, the method comprises administering to the subject naked DNAwhich encodes a peptide, or in another embodiment, two or more peptidesof this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In anotherembodiment, multi-epitope, analogue-based cancer vaccines are utilized(Fikes et al, ibid). Each possibility represents a separate embodimentof the present invention.

Nucleic acids can be administered to a subject via any means as is knownin the art, including parenteral or intravenous administration, or inanother embodiment, by means of a gene gun. In another embodiment, thenucleic acids are administered in a composition, which correspond, inother embodiments, to any embodiment listed herein.

Vectors for use according to methods of this invention can comprise anyvector that facilitates or allows for the expression of a peptide ofthis invention. Vectors comprises, in some embodiments, attenuatedviruses, such as vaccinia or fowlpox, such as described in, e.g., U.S.Pat. No. 4,722,848, incorporated herein by reference. In anotherembodiment, the vector is BCG (Bacille Calmette Guerin), such asdescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vectors useful for therapeutic administration or immunizationof the peptides of the invention, e.g., Salmonella typhi vectors and thelike, will be apparent to those skilled in the art from the descriptionherein.

In another embodiment, the vector further encodes for animmunomodulatory compound, as described herein. In another embodiment,the subject is administered an additional vector encoding same,concurrent, prior to or following administration of the vector encodinga peptide of this invention to the subject.

In another embodiment, the peptides, compositions and vaccines of thisinvention are administered to a subject, or utilized in the methods ofthis invention, in combination with other anti-cancer compounds andchemotherapeutics, including monoclonal antibodies directed againstalternate cancer antigens, or, in another embodiment, epitopes thatconsist of an AA sequence which corresponds to, or in part to, that fromwhich the peptides of this invention are derived.

Various embodiments of dosage ranges are contemplated by this invention.In another embodiment, the dosage is 20 μg per peptide per day. Inanother embodiment, the dosage is 10 μg/peptide/day. In anotherembodiment, the dosage is 30 μg/peptide/day. In another embodiment, thedosage is 40 μg/peptide/day. In another embodiment, the dosage is 60μg/peptide/day. In another embodiment, the dosage is 80 μg/peptide/day.In another embodiment, the dosage is 100 μg/peptide/day. In anotherembodiment, the dosage is 150 μg/peptide/day. In another embodiment, thedosage is 200 μg/peptide/day. In another embodiment, the dosage is 300μg/peptide/day. In another embodiment, the dosage is 400 μg/peptide/day.In another embodiment, the dosage is 600 μg/peptide/day. In anotherembodiment, the dosage is 800 μg/peptide/day. In another embodiment, thedosage is 1000 μg/peptide/day. In another embodiment, the dosage is 1500μg/peptide/day. In another embodiment, the dosage is 2000μg/peptide/day.

In another embodiment, the dosage is 10 μg/peptide/dose. In anotherembodiment, the dosage is 30 μg/peptide/dose. In another embodiment, thedosage is 40 μg/peptide/dose. In another embodiment, the dosage is 60μg/peptide/dose. In another embodiment, the dosage is 80μg/peptide/dose. In another embodiment, the dosage is 100μg/peptide/dose. In another embodiment, the dosage is 150μg/peptide/dose. In another embodiment, the dosage is 200μg/peptide/dose. In another embodiment, the dosage is 300μg/peptide/dose. In another embodiment, the dosage is 400μg/peptide/dose. In another embodiment, the dosage is 600μg/peptide/dose. In another embodiment, the dosage is 800μg/peptide/dose. In another embodiment, the dosage is 1000μg/peptide/dose. In another embodiment, the dosage is 1500μg/peptide/dose. In another embodiment, the dosage is 2000μg/peptide/dose.

In another embodiment, the dosage is 10-20 μg/peptide/dose. In anotherembodiment, the dosage is 20-30 μg/peptide/dose. In another embodiment,the dosage is 20-40 μg/peptide/dose. In another embodiment, the dosageis 30-60 μg/peptide/dose. In another embodiment, the dosage is 40-80μg/peptide/dose. In another embodiment, the dosage is 50-100μg/peptide/dose. In another embodiment, the dosage is 50-150μg/peptide/dose. In another embodiment, the dosage is 100-200μg/peptide/dose. In another embodiment, the dosage is 200-300μg/peptide/dose. In another embodiment, the dosage is 300-400μg/peptide/dose. In another embodiment, the dosage is 400-600μg/peptide/dose. In another embodiment, the dosage is 500-800μg/peptide/dose. In another embodiment, the dosage is 800-1000μg/peptide/dose. In another embodiment, the dosage is 1000-1500μg/peptide/dose. In another embodiment, the dosage is 1500-2000μg/peptide/dose.

In another embodiment, the total amount of peptide per dose or per dayis one of the above amounts. In another embodiment, the total peptidedose per dose is one of the above amounts.

Each of the above doses represents a separate embodiment of the presentinvention.

EXPERIMENTAL DETAILS SECTION Example 1: Binding of HLA-A0201 and -A0301by Synthetic Peptide Analogues Derived from WT1 Materials andExperimental Methods

Peptides

Peptides were synthesized by Genemed Synthesis Inc, CA usingfluorenylmethoxycarbonyl chemistry and solid phase synthesis, and werepurified by high pressure liquid chromatography (HPLC). The quality ofthe peptides was assessed by HPLC analysis, and the expected molecularweight was measured using matrix-assisted laser desorption massspectrometry. Peptides were sterile and >90% pure. The peptides weredissolved in DMSO and diluted in PBS at pH 7.4 or saline solution toyield a concentration of 5 milligrams per milliliter (mg/ml) and werestored at −80° C. For in vitro experiments, an irrelevant controlpeptide, HLA A24 consensus, was used.

Peptide Sequence Analysis

Peptide sequence analysis was performed using 2 databases. The first wasthe software of the Bioinformatics & Molecular Analysis Section(National Institutes of Health, Washington, D.C.) (Parker K C et al,Scheme for ranking potential HLA-A2 binding peptides based onindependent binding of individual peptide side-chains. J Immunol 152:163-175, 1994), which ranks 9-mer or 10-mer peptides on a predictedhalf-time dissociation coefficient from HLA class I molecules. Thesecond database, SYFPEITHI prediction software, is described inRammensee H G et al (SYFPEITHI: database for MHC ligands and peptidemotifs. Immunogenetics 50: 213-219, 1999). Irrelevant control peptidesused for in vitro experiments were: RAS (TEYKLVVVGAPGVGKSALTIQ; SEQ IDNo: 46) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID No: 47) for ClassII, and HIV pol (ILKEPVHGV; SEQ ID No: 48) or CML F (YLKALQRPY; SEQ IDNo: 49) for Class I.

Cell Lines

Cell lines were cultured in RPMI 1640 medium supplemented with 5% FCS,penicillin, streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37° C.in humidified air containing 5% CO₂. T2 is a human cell line lackingTAP1 and TAP2 and therefore unable to present peptides derived fromcytosolic proteins. Raji cells are a human Burkitt lymphoma cells thatexhibit a high level of TAP expression.

Human mesothelioma cell lines studied included: sarcomatoid (VAMT,H2373, H28), epithelioid (H2452) and biphasic (JMN, MSTO and H-Meso1A).Cell lines were obtained from the following sources: H-Meso1A: NCI,Bethesda, Md.; JMN and VAMT: Dr. Sirotnak, Memorial Sloan KetteringCancer Center (MSKCC); H-2452 and H2373: Dr. Pass, Karmanos CancerInstitute, Wayne State University, Detroit, Mich.; H28 and MSTO:American Type Culture Collection (ATCC, Manassas, Va.). Cell lines weremaintained in media recommended by the suppliers and incubated in ahumidified incubator with 5% CO₂.

Mesothelioma cell lines Meso 11, Meso 34, Meso 37, Meso 47 and Meso 56were obtained from Dr. M Gregoire (Institute of Biology, Nantes, France)and cultured in RPMI 1640 (Life Technologies)+10% fetal calf serum(FCS), 1% penicillin-streptomycin, and 1% L-glutamine. All cells wereHLA typed by the Department of Cellular Immunology at MSKCC. Melanomacell line Mewo (WT1⁻ A201⁺) was obtained from the ATCC. SKRC-52 renalcell carcinoma was obtained from L. Old of the Ludwig Institute.Leukemia cell lines were cultured in RPMI 1640+10% FCS, 1%penicillin-streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37°C./5% CO₂. LAMA81, BV173 and 697, Ph⁺ leukemias that are all WT1⁺ andA0201⁺, were provided by Dr. H J Stauss (University College London).SKLY-16 is a human B cell lymphoma (WT1⁻, A0201⁺); K562, RwLeu4 andHL60, all WT1⁺ leukemias, were obtained from the ATCC.

T2 Assay for Peptide Binding and Stabilization of HLA A0201 Molecules

T2 cells (TAP⁻, HLA-A0201⁺) were incubated overnight at 27° C. at aconcentration of 1×10⁶ cells/ml in FCS-free RPMI medium supplementedwith 5 μg/ml human ß₂m (Sigma, St Louis, Mo.) in the absence (negativecontrol) or presence of either a positive reference tyrosinase peptideor test peptides at various final concentrations (50, 10, 1, and 0.1micrograms (μg)/ml). Following a 4-hour incubation with 5 μg/mlbrefeldin A (Sigma), T2 cells were labeled for 30 minutes at 4° C. witha saturating concentration of anti-HLA-A2.1 (BB7.2) mAb, then washedtwice. Cells were then incubated for 30 minutes, 4° C. with a saturatingconcentration of FITC-conjugated goat IgG F(ab′)2 anti-mouse Ig (Caltag,San Francisco, Calif.), washed twice, fixed in PBS/1% paraformaldehydeand analyzed using a FACS Calibur@ cytofluorometer (Becton Dickinson,Immunocytometry Systems, San Jose, Calif.).

The mean intensity of fluorescence (MIF) observed for each peptideconcentration (after dividing by the MIF in the absence of peptide) wasused as an indication of peptide binding and expressed as a“fluorescence index.” Stabilization assays were performed similarly.Following initial evaluation of peptide binding at time 0, cells werewashed in RPMI complete medium to remove free peptides and incubated inthe continuous presence of 0.5 μg/ml brefeldin-A for 2, 4, 6 or 8 hours.

The number of stable peptide-HLA-A2.1 complexes was estimated asdescribed above by immunofluorescence. The half time of complexes is anestimate of the time required for a 50% reduction of the MIF value attime=0.

Results

Peptides having predicted affinity for HLA-A0201 and HLA-A0301 moleculeswere identified from the WT1 sequence. These WT-1 native peptides weremodified to generate heteroclitic peptides with increased predictedbinding to HLA-A0201 and HLA-A0301 molecules, as shown in Tables 1-2.Several of the heteroclitic peptides significantly stabilized HLA-A0201and HLA-A0301 molecules in thermostabilization assays using a TAP1/2negative cell line (T2) and Raji HLA-A0301 cells. Specifically, WT1-A1,B1, and C1 exhibited similar or increased binding compared to thecorresponding native peptides WT1-A, B, and C. WT1-D1 exhibited similaror increased binding compared to corresponding native peptide WT1-D(FIG. 1A). A comparison of HLA A0301 binding of A3 WT1-A, -B, -C, and -Dwith each of their respective three analogues demonstrated similarbinding (FIGS. 1B-5E).

Thus, heteroclitic WT1 peptides of the present invention exhibitenhanced binding to HLA class I molecules.

TABLE 1 HLA 0201-binding native peptides from WT-1 andsynthetic analogues BIMAS Name Sequence SEQ ID NO: score WT-1 A (native)RMFPNAPYL  5  313 WT-1 A1 (ANALOGUE) Y MFPNAPYL  6 1444 WT-1 B (native)SLGEQQYSV  7  285 WT-1 B1 (ANALOGUE) Y LGEQQYSV  8 1311 WT-1 C (native)ALLPAVPSL  9  181 WT-1 C1 (ANALOGUE) Y LLPAVPSL 10  836 WT-1 D (native)NLGATLKGV 11  159 WT-1 D1 (ANALOGUE) Y LGATLKGV 12  735 WT-1 E (native)DLNALLPAV 13   11 WT-1 E1 (ANALOGUE) Y LNALLPAV 14  735 WT-1 F (native)GVFRGIQDV 15   51 WT-1 F1 (ANALOGUE) G LR RGIQDV 16  591 WT-1 G (native)KRYFKLSHL 17    1 WT-1 G1 (ANALOGUE) K L YFKLSHL 18  550 WT-1 H (native)ALLLRTPYS 19    1 WT-1 H1 (ANALOGUE) ALLLRTPY V 20 1415 WT-1 J (native)CMTWNQMNL 21   15 WT-1 J1 (ANALOGUE) Y MTWNQMNL 22   70

TABLE 2 HLA 0201-binding native peptides from WT-1 andsynthetic analogues SEQ ID BIMAS Name Sequence NO: scoreA3 WT-1 A (native) NMHQRNMTK 23  40 A3 WT-1 A1 (ANALOGUE) NM Y QRNMTK 24200 A3WT-1 A2 (ANALOGUE) NMHQR V MTK 25 120 A3 WT-1 A3 (ANALOGUE) NM YQR V MTK 26 600 A3 WT-1 B (native) QMNLGATLK 27  20 A3WT-1 B1 (ANALOGUE)QM Y LGATLK 28 100 A3WT-1 B2 (ANALOGUE) QMNLG V TLK 29  60A3WT-1 B3 (ANALOGUE) QM Y LG V TLK 30 300 A3WT-1 C (native) FMCAYPGCNK31  30 A3WT-1 C1 (ANALOGUE) FM Y AYPGCNK 32 150 A3 WT-1 C2 (ANALOGUE)FMCAYP F CNK 33  90 A3 WT-1 C3 (ANALOGUE) FM Y AYP F CNK 34 450A3WT-1 D (native) KLSHLQMHSR 35  18 A3WT-1 D1 (ANALOGUE) KL Y HLQMHSR 36 90 A3 WT-1 D2 (ANALOGUE) KLSHLQMHS K 37  90 A3 WT-1 D3 (ANALOGUE) KL YHLQMHS K 38 450

Example 2: Induction of Immune Responses Against Synthetic PeptideAnalogues Derived from WT1 Materials and Experimental Methods

Peptide Stimulations

PBMC were purified from HLA-A0201 positive healthy donors and CMLpatients by centrifugation in Ficoll-Paque centrifugation medium(Amersham Biosciences). Peripheral blood dendritic cells (DC) weregenerated as follows: Monocyte-enriched PBMC fractions were isolated,using a plastic adherence technique, from total PBMC. Plastic-adherentcells were cultured further in RPMI 1640 medium (Invitrogen) with 1-5%autologous plasma, 1000 units per milliliter (U/mL) recombinant humaninterleukin (IL)-4 (Schering-Plough, N.J.), and 1000 U/mL recombinanthuman granulocyte-macrophage colony-stimulating factor (GM-CSF)(Immunex, Seattle).

On days 2 and 4 of incubation, fresh culture medium supplemented withIL-4 and GM-CSF was added. On day 6, half of the medium was exchangedfor culture medium containing IL-4, GM-CSF, 10 ng/mL recombinant humantumor necrosis factor (TNF)-alpha (R&D system) and 500 ng/ml trimericsoluble CD40L (Immunex, Seattle). On day 9, cells were harvested andused as APC for antigen stimulation. The cells expressed DC-associatedantigens, such as CD80, CD83, CD86, and HLA class I and class II ontheir cell surfaces.

T lymphocytes were isolated from the same donors by use of negativeselection by depletion with an anti-CD11b, anti-CD56 and CD19 monoclonalantibody (Miltenyi, Calif.). 1×10{circumflex over ( )}6 T lymphocyteswere cultured with 1×10{circumflex over ( )}5 autologous DC in RPMI 1640containing 5% heat-inactivated human autologous plasma with 10 μg/mLpeptide and 2 μg/ml β₂ microglobulin, 5 ng/mL recombinant human IL-7(Genzyme), and 0.1 ng/ml IL-12 in 24 well plates.

After culture for 3 days, 20 U/ml of recombinant IL-2 (SandozPharmaceutical) was added. After 10 days, 1×10{circumflex over ( )}6cells were stimulated again by adding 2×10{circumflex over ( )}5autologous magnetically isolated CD14⁺ monocytes together with 10 ng/mlIL-7, 20 U/ml IL-2, and 10 μg/mL peptide. In some cases, after culturefor another 7 days, the cells were stimulated a third time in the samemanner. After the last stimulation, CD8⁺ T cells were isolatedmagnetically, and cytotoxicity and gamma-IFN secretion were determined.

Results

To determine the ability of heteroclitic WT1 peptides to generate immuneresponses against native and heteroclitic WT peptides, the CD3⁺ PBMCsubpopulation of a healthy donor was isolated and stimulated withautologous monocyte-derived, peptide-pulsed DC, then re-stimulated withpeptide-pulsed CD14⁺ monocytes. The presence of activated,antigen-specific T cells was then determined using pulsed, HLA-matchedleukemic cell lines. Several analogue peptides generated greater immuneresponses (i.e. increased T cell precursor frequency, in comparison withthe native peptides) by IFN gamma ELISPOT (FIG. 2A) and chromium releaseassay (FIG. 2B). Similar results were observed using CD3⁺ (FIGS. 3B-D)and CD8⁺ (FIG. 3A) subpopulations of donors. Moreover, CD8⁺ T cellsstimulated with the heteroclitic WT1 peptides cross-reacted with thenative WT1 peptides and were able to lyse HLA-matched CML blasts (FIGS.4A-B).

Thus, heteroclitic WT1 peptides of the present invention are able togenerate T cells that (a) secrete inflammatory cytokines and (b) performcytolysis in response to cells presenting WT1 peptides. In addition, theT cells generated by the heteroclitic WT1 peptides recognize both nativeand heteroclitic WT1 peptides

Example 3: Selection of Synthetic WT1 Peptides that Bind HLA Class IIMolecules

In order to identify WT1 peptides that bind to many different HLA classII molecules with relatively high affinities, allele frequencies ofHLA-DRB in the North American Caucasian population were determined,using the NCBI MHC database (Wheeler D L et al, Database resources ofthe National Center for Biotechnology Information. Nucleic Acids Res.2005 Jan. 1; 33:D39-45; Wheeler D L et al, Database resources of theNational Center for Biotechnology Information. Nucleic Acids Res. 2006Jan. 1; 34:D173-80). Using the SYFPEITHI epitope prediction algorithm, 2peptides predicted to bind HLA-DRB molecules with relatively highaffinities were identified from WT1 (Table 3).

TABLE 3 WT1 native peptides predicted binding to HLA-DR alleles based onSYFPEITHI algorithm (0 (low)-28 (high)). DRB DRB DRB DRB DRB DRB Peptideidentifier SEQ ID No: 101 301 401 701 1101 1501 Allele frequency 17.9%18.6% 13.8% 25.5% 10.4% 15.9% 427 1 15 7 12 8 7 4 423 2 15 17 20 14 1024 331 3 28 2 28 18 25 10 328 4 28 11 28 18 25 20

AA sequences of the peptides in Table 3 are LVRHHNMHQRNMTKL (427);RSDELVRHHNMHQRNMTKL (423); NKRYFKLSHLQMHSR (331); andPGCNKRYFKLSHLQMHSRKHTG (328).

Thus, HLA class II-binding WT1 peptides of the present invention bind toHLA class II molecules in a large percentage of the population.

Example 4: HLA Class II Molecule-Binding, WT1 Peptides Stimulate CD4⁺ TCells Materials and Experimental Methods (this and Subsequent Examples)

Preparation of DC and CD4⁺ Effector Cells

PBMC were Ficoll-purified from blood and resuspended at 5×10{circumflexover ( )}6/ml in Ex-Vivo-15® medium (BioWhittaker, Walkersville, Md.)containing 1% autologous plasma. After a 2-hour incubation at 37° C.,the non-adherent fraction was harvested and washed repeatedly with PBS,then resuspended in media containing 1×10³ IU/ml GM-CSF and 0.0032 IU/mlIL-4. On day 2 and 4, the same media was added as re-feed (i.e., ½ thevolume of media, containing enough cytokines for the entire dish, wasadded). On day 5, 10 μg/ml of peptide was added.

On day 6, a maturation cocktail of cytokines was added, and cells werecultured for another 48 hours. The maturation cocktail consisted of:4×10² IU/ml IL-1-beta, 0.0032 IU/ml IL-4, 1×10³ IU/ml IL-6, 1×10³ IU/mlGMCSF, 10 μg/ml TNF-alpha, and 1 μg/ml PGE2.

On day 7, DC were harvested and washed twice with RPMI, counted,aliquoted and resuspended at 1×10⁶/ml in X-vivo 15® media (withoutserum). Peptides were added to a final concentration of 10 μg/ml, andincubated for 2 h, 37° C. and 5% CO2, gently re-suspending every 15minutes, then washed twice in HBSS and re-suspended in RPMI+5%autologous plasma at an appropriate concentration depending on thenumber of effectors isolated in the next step.

In addition, on day 7, additional PBMC were used to generate additionalDC and CD3⁺ cells. DC were isolated from the adherent fraction andprepared as described above for the second stimulation of the effectorcells on day 14. CD3⁺ cells were isolated from the non-adherent fractionby negative selection and stimulated with the previously prepared DC byre-suspending the CD3⁺ cells at a concentration of 2×10⁶ cells/ml inRPMI+5% autologous plasma, and adding DC at an effector:DC ratio of 20:1and 10 ng/ml IL-15. Cells were then plated in 2 ml and co-incubated at37° C. and 5% CO₂ for 1 week.

On day 14, the CD3⁺ cells were stimulated a second time with the secondbatch of DC in the same manner, except that 1×10⁶ cells/ml were mixedwith DC at an effector:DC ratio of 50:1. On day 18, the same media wasadded as re-feed. On day 20, the DC from the previous generation weredefrosted and incubated in maturation cytokines in X-vivo 15@ media. Onday 21, the ELISPOT assay was conducted.

ELISPOT Assay

Plates were pre-wet with 30 μl/well 70% alcohol, shaking the plates toensure coverage of the entire surface area, washed 3 times with 150μl/well sterile PBS, then incubated overnight at 4° C. with 10 μg/mlcoating antibody (anti-INF clone) in PBS, 100 μl/well, wrapped inaluminum foil. Plates were then washed 2 times with 150 μl/well PBS and1 time with RPMI/10% autologous plasma (AP), then blocked with 150μl/well RPMI/5% AP for 2 hours at 37° C. PBMC were suspended in RPMI/5%AP at 1×10⁶/ml. 1×10⁵ cells and 2 μg of the appropriate peptides wereadded per well, and the volume brought up to 200 μl/well with media. 1μl/well of 2.5 mg/ml stock of PHA was added to the control wells. Plateswere wrapped in aluminum foil and incubated for 20 hours at 37° C.

To develop, plates were washed 3 times with PBS/0.05% Tween 2 and 3times with PBS. 100 μl/well anti-INF-gamma-Biotin (Clone 7-B6-1),diluted 1:500 in PBS/0.5% BSA, was added, and plates were incubated for2 hours at 37° C. After 1 hour and 30 minutes, Avidin-peroxidase Complex(ABC) (Vectastain Elite Kit, Vector) was prepared by adding 1 drop ofreagent A and 1 drop of reagent B to 10 ml of PBS/0.1% Tween20, and wasstored at room temperature (rt) wrapped in aluminum foil. Plates werewashed 3 times with PBS/0.05% Tween and 3 times with PBS, then 100μl/well of Avidin-peroxidase Complex was added and plates incubated for1 hour at rt wrapped in aluminum foil, then washed 3 times withPBS/0.05% Tween-20, followed by 3 times with PBS. 100 μl/well ofsubstrate was added, plates were incubated for 4 minutes at rt in thedark, and the reaction was stopped with water. Wells were dried andplates stored overnight in the dark at rt. Spot numbers wereautomatically determined with the use of a computer-assisted video imageanalyzer with KS ELISPOT 4.0 software (Carl Zeiss Vision, Germany).

Preparation of Substrate

To prepare solution #1: (acetate buffer), 23.4 ml dd H₂O, 2.3 ml 0.1 NAcetic Acid, and 5.5 ml 0.1N Sodium Acetate were mixed. To preparedsolution #2, 1 tablet of AEC (Sigma) was dissolved in 2.5 ml ofdimethylformamide. Then 1.25 ml of solution #2 was mixed with 23.7 ml ofsolution #1, 13 μl of 30% H₂O₂ was added, and the resulting solutionmixed well and filtered using a 0.45 μm filter.

Cross Priming Experiments

A CD3⁺ in vitro stimulation was performed as described above. 2×10⁶immature DCs were incubated with total cellular lysate from 2×10⁶ tumorcells that was previously prepared by 3 freeze/thaw cycles. Following an18 hour incubation, maturation cytokines were added to the DCs asdescribed above. CD3⁺ cells were stimulated 3 times with theseautologous mature DCs, after which T cells were tested in an IFN-gammaELISPOT assay for reactivity against autologous, mature DCs that hadbeen pulsed with individual CD4⁺ peptides when in the immature state.These DCs were exposed to peptide again during the ELISPOT assay asdescribed above.

Chromium 51 Cytotoxicity Assay

The presence of specific CTL was measured in a standard 4 h-chromiumrelease assay. Target cells were pulsed with 10 microgram (mcg)/ml ofsynthetic peptides overnight at 37° C. and labeled with 300 μCi of Na₂⁵¹CrO₄ (NEN Life Science Products, Inc., Boston, Mass.). After extensivewashing, target cells were incubated with T cells at an E:T ratioranging from 100:1 to 10:1. All conditions were performed in triplicate.Plates were incubated for 4 hours at 37° C. in 5% CO₂. Supernatantfluids were harvested and radioactivity was measured in a gamma counter.Percent specific lysis was determined from the following formula:100×[(experimental release minus spontaneous release)/(maximum releaseminus spontaneous release)]. Maximum release was determined by lysis ofradiolabeled targets in 2.5% Triton X-100.

Statistics

Statistical analyses were performed on Statview software (SAS Institute,Cary, N.C.) using a two-tailed unpaired t-test, with the level ofstatistical significance set at 0.05.

Results

To determine the ability of the HLA class II-binding WT1 peptides of thepresent invention to stimulate CD4⁺ T cells, CD4⁺ PBMC subpopulations ofhealthy donors were isolated and stimulated with autologousmonocyte-derived, peptide-pulsed DC, then re-stimulated withpeptide-pulsed CD14⁺ monocytes. Peptide 328, and to a slightly lessextent peptide 423, stimulated a significant peptide-specific CD4+ Tcell response in a variety of donors with different HLA-DRB1 types, asshown by IFN-γ ELISPOT (FIG. 5 ). As expected, cells stimulated with RAS(irrelevant control peptide) or with APC alone did not produce IFN-γover background levels.

Thus, HLA class II-binding WT1 peptides of the present invention areable to stimulate T cells that recognize cells presenting WT1 peptides.

Example 5: WT1-Expressing Cells Process and Present Peptides of thePresent Invention

Cross-priming studies were performed to determine whether WT1-expressingcells process and present peptides of the present invention or thecorresponding native peptides. Total tumor lysates were prepared from 3different cell lines: 697 (WT1⁺, HLA A0201⁺), an e1a2 leukemia cellline; JMN (WT1⁺, HLA A0201⁺) a biphasic mesothelioma cell line, and as acontrol, MeWo (WT1⁻, HLA A0201⁺), a malignant melanoma cell line. DCsfrom healthy A0201⁺ donors were incubated for 18 hours with the tumorlysates and used to stimulate autologous CD3⁺ T cells. Following 3stimulations, the T cells were tested for their reactivity to autologousDCs pulsed with the WT1 peptides. T cells that had been stimulated withWT1⁺ tumor lysates recognized the individual HLA class II peptides (FIG.6A-B), while T cells stimulated by DCs pulsed with MeWo lysate did notstimulate WT1-specific T cells. As a positive control, 697 lysate wasused in the ELISPOT; this yielded spot numbers approximately equal to423 and 328. These experiments were repeated in 5 separate donors.Stimulated T cells recognized WT1DR peptide 328 in 3/5 experiments andWT1DR 427 in all experiments. Therefore, despite the low expression ofWT1 transcript in the mesothelioma cell lines (see below), WT1 CD4epitopes of the present invention were processed and presented by HLAclass II molecules of mesothelioma cells.

These findings show that peptides of the present invention are (a) takenup and presented by APC in an antigenic form; and (b) are presented byAPC exposed to WT1-expressing tumor cells; and (c) APC exposed to WT1122 and 122A1 peptides elicit the formation of T cells that recognizeWT1-expressing tumor cells. Thus, WT1-expressing cells, includingmesothelioma and leukemia cells, process and present peptides of thepresent invention or the corresponding native peptides.

Example 6: Antigen-Specific CD4⁺ T Cells Generated by Peptides of thePresent Invention Recognize WT1-Expressing Tumor Cells

To test whether antigen-specific CD4⁺ T cells generated by peptides ofthe present invention recognize WT1-expressing tumor cells,peptide-stimulated T cells were challenged in an IFN-gamma ELISPOT withWT-1⁺ and -negative tumor cells. A sufficient amount of WT1 peptide waspresented on the surface of the WT1⁺ mesothelioma tumor cell for T cellsstimulated with individual WT1DR peptides to recognize mesotheliomatumor cells, compared to the control WT1 negative melanoma cells (FIG. 7). Thus, vaccination with peptides of the present invention results ingeneration of antigen-specific T cells with activity againstWT1-expressing tumors.

Example 7: WT1 Expression in Human Mesothelioma Cell Lines Materials andExperimental Methods

Quantitative RT-PCR for WT-1 Transcripts

Total RNA was isolated from cell lines by phenol/chloroform extraction.RNA purity was confirmed by absorbance at 260 nm. The RT reaction wasadapted from protocols supplied by Applied Biosystems (Foster City,Calif.). Beginning with 1 mcg total RNA, random hexamers and reversetranscriptase were used to isolate cDNA. For the PCR reaction, cDNA wasmixed with the following WT1 primers and probe: forward primer (locatedon exon 7): 5′ CAGGCTGCAATAAGAGATATITTAAGCT-3′ (SEQ ID No: 39); andreverse primer (located on exon 8): 5′-GAAGTCACACTGGTATGGTTTCTCA-3′ (SEQID No: 40); Taqman probe (located on exon 7)5′-CTTACAGATGCACAGCAGGAAGCACACTG-3′ (SEQ ID No: 41). The fluorescent WT1probe 5′-56-FAM/CTTACAGATGCACAGCAGGAAGCACACTG/3BHQ_1/-3 (SEQ ID No: 42)was labeled with 6-carboxyfluorescein phosphoramide (FAM) as reporterdye at the 5′-end and with the quencher dye carboxytetramethylrhodamine(TAMRA) at the 3′-end (Integrated DNA Technologies, Coralville, Iowa).The parameters for the PCR reaction were: 2 minutes at 50° C., 10 min at95° C.; followed by 50 cycles of 15 s at 95° C. and 60 s at 62° C. Eachreaction was performed in triplicate, and discrepancies >1 Ct in 1 ofthe wells were excluded. The Q-RT-PCR reaction and fluorescencemeasurements were made on the Applied Biosystems 7500 Real Time® PCRSystem. Control ABL primers and probes were: forward5′-TGGAGATAACACTCTAAGCATAACTAAAGGT-3 (SEQ ID No: 43; located onEnF-10030)′; reverse 5′-GATGTAGTTGCTTGGGACCCA-3′ (SEQ ID No: 44; locatedon ENR-1063); fluorescent probe 5′-/56FAM/CCATTTTGGTTTGGGCTTCACACCATT/3BHQ_1/-3′ (SEQ ID No: 45; located onENPr-1043).

Results

To determine WT1 expression levels in mesothelioma, WT1 transcriptlevels in a number of human mesothelioma cell lines (sarcomatoid,epitheloid and biphasic) were quantified by RT-PCR and compared tovarious leukemia cell lines with known WT1 expression. 12/12mesothelioma cell lines expressed WT1 message, in most cases at a lowerlevel than leukemic cell lines (FIG. 8 ). By contrast, melanoma (MeWo)and lymphoma (SKLY16) cell lines were WT1 negative. SK-RC-52, a humanrenal cell carcinoma cell line did not express WT1, despite the lowexpression of WT1 in adult renal podocytes. Flow cytometry analysisconfirmed that all the mesothelioma cell lines expressed class IImolecules, and some (JMN and H-2452) expressed class I molecules.

Thus, methods of the present invention can be used to induce immuneresponses and vaccination against mesothelioma cells.

What is claimed is:
 1. An isolated Wilms' tumor 1 (WT1) peptide linkedto another molecule, wherein the peptide consists of the amino acidsequence RSDELVRHHNMHQRNMTKL (SEQ ID NO: 2), or an isolated W I 1peptide linked to another molecule, wherein the peptide consists of theamino acid sequence PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 4), or thecombination of an isolated WT1 peptide linked to another molecule,wherein the peptide consists of the amino acid sequenceRSDELVRHHNMHQRNMTKL (SEQ ID NO: 2), and an isolated WT1 peptide linkedto another molecule, wherein the peptide consists of the amino acidsequence PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 4).
 2. The isolated WT1peptide linked to another molecule of claim 1 wherein the moleculelinked to the WT1 peptide is an immunomodulating compound.
 3. Theisolated WT1 peptide linked to another molecule of claim 2 wherein theimmunomodulating compound is a cytokine, chemokine, or complementcomponent that enhances expression of immune system accessory oradhesion molecules, their receptors, or combinations thereof.
 4. Theisolated WT1 peptide linked to another molecule of claim 2 wherein theimmunomodulating compound is an interleukin, interferon alpha, beta orgamma, tumour necrosis factor, granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF),granulocyte colony stimulating factor (G-CSF), a chemokine, a complementcomponent, or combinations thereof.
 5. The isolated WT1 peptide linkedto another molecule of claim 2 wherein the immunomodulating compoundstimulates expression, or enhances expression of OX40, OX40L (gp34),lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-2, ICAM-3, cytokinereceptors, CD28, CTLA-4, a B7 molecule, a heat stable antigen (HSA),chondroitin sulfate-modified MHC invariant chain (Ii-CS), or anintracellular adhesion molecule 1 (ICAM-1), or combinations thereof. 6.The combination of the isolated WT1 peptide linked to another moleculeof claim 1, wherein the peptide consists of the peptide having the aminoacid sequence PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 4), and the isolatedWT1 peptide linked to another molecule, wherein the peptide consists ofthe peptide having the amino acid sequence RSDELVRHHNMHQRNMTKL (SEQ IDNO: 2).
 7. The combination of claim 6, further comprising an isolatedWT1 peptide linked to another molecule wherein the peptide consists ofthe peptide having the amino acid sequence YMFPNAPYL (SEQ ID NO:6).
 8. Acomposition comprising the isolated WT1 peptide linked to anothermolecule of claim 1, and an immunomodulating compound.
 9. Thecomposition of claim 8 wherein the immunomodulating compound is acytokine, chemokine, or complement component that enhances expression ofimmune system accessory or adhesion molecules, their receptors, orcombinations thereof.
 10. The composition of claim 8 wherein theimmunomodulating compound is an interleukin, interferon alpha, beta orgamma, tumour necrosis factor, granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF),granulocyte colony stimulating factor (G-CSF), a chemokine, a complementcomponent, or combinations thereof.
 11. The composition of claim 8wherein the immunomodulating compound stimulates expression, or enhancesexpression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2,TRAP, ICAM-2, ICAM-3, cytokine receptors, CD28, CTLA-4, a B7 molecule, aheat stable antigen (HSA), chondroitin sulfate-modified MHC invariantchain (Ii-CS), or an intracellular adhesion molecule 1 (ICAM-1), orcombinations thereof.
 12. The isolated WT1 peptide linked to anothermolecule of claim 1, wherein the molecule linked to the WT1 peptide is aprotein or a polypeptide.
 13. The isolated WT1 peptide linked to anothermolecule of claim 1, wherein the WT1 peptide linked to another moleculeis by a peptide bond, an ester or an ether.
 14. The isolated WT1 peptidelinked to another molecule of claim 1, wherein the WT1 peptide is linkedto the molecule through a homo-bifunctional agent or ahetero-bifunctional agent.
 15. An isolated WT1 peptide linked to anothermolecule, wherein the peptide comprises the peptide having the aminoacid sequence PGCNKRYFKLSHLQMHSRKHTG (SEQ ID No: 4) and the peptidehaving the amino acid sequence RSDELVRHHNMHQRNMTKL (SEQ ID No: 2), andfurther comprises a peptide having the amino acid sequence YMFPNAPYL(SEQ ID NO:6).
 16. The isolated WT1 peptide linked to another moleculeof claim 15, wherein the molecule linked to the WT1 peptide is a proteinor a polypeptide.
 17. The isolated WT1 peptide linked to anothermolecule of claim 15, wherein the molecule linked to the WT1 peptide isan immunomodulating compound.